Bivalent, bispecific antibodies

ABSTRACT

The present invention relates to novel domain exchanged, bivalent, bispecific antibodies, their manufacture and use.

PRIORITY TO RELATED APPLICATION(S)

This application claims the benefit of European Patent Application No.07024867.9, filed Dec. 21, 2007, which is hereby incorporated byreference in its entirety.

The present invention relates to novel bivalent, bispecific antibodies,their manufacture and use.

BACKGROUND OF THE INVENTION

Engineered proteins, such as bi- or multispecific antibodies capable ofbinding two or more antigens are known in the art. Such multispecificbinding proteins can be generated using cell fusion, chemicalconjugation, or recombinant DNA techniques.

A wide variety of recombinant bispecific antibody formats have beendeveloped in the recent past, e.g. tetravalent bispecific antibodies byfusion of, e.g. an IgG antibody format and single chain domains (seee.g. Coloma, M. J., et. al., Nature Biotech. 15 (1997) 159-163; WO2001077342; and Morrison, S., L., Nature Biotech. 25 (2007) 1233-1234.

Also several other new formats wherein the antibody core structure (IgA,IgD, IgE, IgG or IgM) is no longer retained such as dia-, tria- ortetrabodies, minibodies, several single chain formats (scFv, Bis-scFv),which are capable of binding two or more antigens, have been developed(Holliger, P., et. al, Nature Biotech 23 (2005) 1126-1136; Fischer, N.,and Léger, O., Pathobiology 74 (2007) 3-14; Shen, J., et. al., J.Immunol. Methods 318 (2007) 65-74; Wu, C., et al., Nature Biotech 25(2007) 1290-1297).

All such formats use linkers either to fuse the antibody core (IgA, IgD,IgE, IgG or IgM) to a further binding protein (e.g. scFv) or to fusee.g. two Fab fragments or scFv (Fischer, N., and Léger, O., Pathobiology74 (2007) 3-14). While it is obvious that linkers have advantages forthe engineering of bispecific antibodies, they may also cause problemsin therapeutic settings. Indeed, these foreign peptides might elicit animmune response against the linker itself or the junction between theprotein and the linker. Further more, the flexible nature of thesepeptides makes them more prone to proteolytic cleavage, potentiallyleading to poor antibody stability, aggregation and increasedimmunogenicity. In addition one may want to retain effector functions,such as e.g. complement-dependent cytotoxicity (CDC) or antibodydependent cellular cytotoxicity (ADCC), which are mediated through theFcpart by maintaining a high degree of similarity to naturally occurringantibodies.

Thus ideally, one should aim at developing bispecific antibodies thatare very similar in general structure to naturally occurring antibodies(like IgA, IgD, IgE, IgG or IgM) with minimal deviation from humansequences.

In one approach bispecific antibodies that are very similar to naturalantibodies have been produced using the quadroma technology (seeMilstein, C. and A. C. Cuello, Nature, 305 (1983) 537-40) based on thesomatic fusion of two different hybridoma cell lines expressing murinemonoclonal antibodies with the desired specificities of the bispecificantibody. Because of the random pairing of two different antibody heavyand light chains within the resulting hybrid-hybridoma (or quadroma)cell line, up to ten different antibody species are generated of whichonly one is the desired, functional bispecific antibody. Due to thepresence of mispaired byproducts, and significantly reduced productionyields, means sophisticated purification procedures are required (seee.g. Morrison, S. L., Nature Biotech 25 (2007) 1233-1234). In generalthe same problem of mispaired byproducts remains if recombinantexpression techniques are used.

An approach to circumvent the problem of mispaired byproducts, which isknown as ‘knobs-into-holes’, aims at forcing the pairing of twodifferent antibody heavy chains by introducing mutations into the CH3domains to modify the contact interface. On one chain bulky amino acidswere replaced by amino acids with short side chains to create a ‘hole’.Conversely, amino acids with large side chains were introduced into theother CH3 domain, to create a ‘knob’. By coexpressing these two heavychains (and two identical light chains, which have to be appropriate forboth heavy chains), high yields of heterodimer formation (‘knob-hole’)versus homodimer formation (‘hole-hole’ or ‘knob-knob’) was observed(Ridgway, J. B., Protein Eng. 9 (1996) 617-621; and WO 96/027011). Thepercentage of heterodimer could be further increased by remodeling theinteraction surfaces of the two CH3 domains using a phage displayapproach and the introduction of a disulfide bridge to stabilize theheterodimers (Merchant A. M, et al., Nature Biotech 16 (1998) 677-681;Atwell, S., et al., J. Mol. Biol. 270 (1997) 26-35). New approaches forthe knobs-into-holes technology are described in e.g. in EP 1870459A1.Although this format appears very attractive, no data describingprogression towards the clinic are currently available. One importantconstraint of this strategy is that the light chains of the two parentantibodies have to be identical to prevent mispairing and formation ofinactive molecules. Thus this technique is not appropriate for easilydeveloping recombinant, bivalent, bispecific antibodies against twoantigens starting from two antibodies against the first and the secondantigen, as either the heavy chains of these antibodies an/or theidentical light chains have to be optimized.

WO 2006/093794 relates to heterodimeric protein binding compositions. WO99/37791 describes multipurpose antibody derivatives. Morrison et al theJ. Immunolog, 160 (1998) 2802-2808 refers to the influence of variableregion domain exchange on the functional properties of IgG.

SUMMARY OF THE INVENTION

The invention relates to a bivalent, bispecific antibody, comprising:

a) the light chain and heavy chain of an antibody specifically bindingto a first antigen; and

b) the light chain and heavy chain of an antibody specifically bindingto a second antigen,

wherein the variable domains VL and VH are replaced by each other,

and

wherein the constant domains CL and CH1 are replaced by each other.

A further embodiment of the invention is a method for the preparation ofan a bivalent, bispecific antibody according to the invention

comprising the steps of

a) transforming a host cell with

vectors comprising nucleic acid molecules encoding the light chain andheavy chain of an antibody specifically binding to a first antigen, and

vectors comprising nucleic acid molecules encoding the light chain andheavy chain of an antibody specifically binding to a second antigen,

wherein the variable domains VL and VH are replaced by each other,

and

wherein the constant domains CL and CH1 are replaced by each other;

b) culturing the host cell under conditions that allow synthesis of saidantibody molecule; and

c) recovering said antibody molecule from said culture.

A further embodiment of the invention is a host cell comprising

vectors comprising nucleic acid molecules encoding the light chain andheavy chain of an antibody specifically binding to a first antigen, and

vectors comprising nucleic acid molecules encoding the light chain andheavy chain of an antibody specifically binding to a second antigen,

wherein the variable domains VL and VH are replaced by each other,

and

wherein the constant domains CL and CH1 are replaced by each other.

A further embodiment of the invention is a composition, preferably apharmaceutical or a diagnostic composition of the antibody according tothe invention.

A further embodiment of the invention is a pharmaceutical compositioncomprising an antibody according to the invention and at least onepharmaceutically acceptable excipient.

A further embodiment of the invention is a method for the treatment of apatient in need of therapy, characterized by administering to thepatient a therapeutically effective amount of an antibody according tothe invention.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to a bivalent, bispecific antibody, comprising:

a) the light chain and heavy chain of an antibody specifically bindingto a first antigen; and

b) the light chain and heavy chain of an antibody specifically bindingto a second antigen,

wherein the variable domains VL and VH are replaced by each other,

and

wherein the constant domains CL and CH1 are replaced by each other.

Therefore said bivalent, bispecific antibody, comprises:

a) a first light chain and a first heavy chain of an antibodyspecifically binding to a first antigen; and

b) a second light chain and a second heavy chain of an antibodyspecifically binding to a second antigen,

wherein the variable domains VL and VH of the second light chain and thesecond heavy chain are replaced by each other,

and

wherein the constant domains CL and CH1 of the second light chain andthe second heavy chain are replaced by each other.

Thus for said antibody specifically binding to a second antigen thefollowing applies:

within the light chain the variable light chain domain VL is replaced bythe variable heavy chain domain VH of said antibody, and the constantlight chain domain CL is replaced by the constant heavy chain domain CH1of said antibody;

and within the heavy chain the variable heavy chain domain VH isreplaced by the variable light chain domain VL of said antibody, and theconstant heavy chain domain CH1 is replaced by the constant light chaindomain CL of said antibody.

The term “antibody” as used herein refers to whole, monoclonalantibodies. Such whole antibodies consist of two pairs of a “lightchain” (LC) and a “heavy chain” (HC) (such light chain (LC)/heavy chainpairs are abbreviated herein as LC/HC). The light chains and heavychains of such antibodies are polypeptides consisting of severaldomains. In a whole antibody, each heavy chain comprises a heavy chainvariable region (abbreviated herein as HCVR or VH) and a heavy chainconstant region. The heavy chain constant region comprises the heavychain constant domains CH1, CH2 and CH3 (antibody classes IgA, IgD, andIgG) and optionally the heavy chain constant domain CH4 (antibodyclasses IgE and IgM). Each light chain comprises a light chain variabledomain VL and a light chain constant domain CL. The structure of onenaturally occurring whole antibody, the IgG antibody, is shown e.g. inFIG. 1. The variable domains VH and VL can be further subdivided intoregions of hypervariability, termed complementarity determining regions(CDR), interspersed with regions that are more conserved, termedframework regions (FR). Each VH and VL is composed of three CDRs andfour FRs, arranged from amino-terminus to carboxy-terminus in thefollowing order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 ((Janeway, C. A.,Jr. et al., Immunobiology, 5th ed., Garland Publishing (2001); and WoofJ. Burton D Nat Rev Immunol 4 (2004) 89-99). The two pairs of heavychain and light chain (HC/LC) are capable of specifically binding tosame antigen. Thus said whole antibody is a bivalent, monospecificantibody. Such “antibodies” include e.g. mouse antibodies, humanantibodies, chimeric antibodies, humanized antibodies and geneticallyengineered antibodies (variant or mutant antibodies) as long as theircharacteristic properties are retained. Especially preferred are humanor humanized antibodies, especially as recombinant human or humanizedantibodies.

There are five types of mammalian antibody heavy chains denoted by theGreek letters: α, δ, ε, γ, and μ (Janeway, C. A., Jr., et al.,Immunobiology, 5th ed., Garland Publishing (2001)). The type of heavychain present defines the class of antibody; these chains are found inIgA, IgD, IgE, IgG, and IgM antibodies, respectively (Rhoades R A,Pflanzer R G (2002). Human Physiology, 4th ed., Thomson Learning).Distinct heavy chains differ in size and composition; α and γ containapproximately 450 amino acids, while μ and ε have approximately 550amino acids.

Each heavy chain has two regions, the constant region and the variableregion. The constant region is identical in all antibodies of the sameisotype, but differs in antibodies of different isotype. Heavy chains γ,α and δ have a constant region composed of three constant domains CH1,CH2, and CH3 (in a line), and a hinge region for added flexibility(Woof, J., Burton D Nat Rev Immunol 4 (2004) 89-99); heavy chains μ andε have a constant region composed of four constant domains CH1, CH2,CH3, and CH4 (Janeway, C. A., Jr., et al., Immunobiology, 5th ed.,Garland Publishing (2001)). The variable region of the heavy chaindiffers in antibodies produced by different B cells, but is the same forall antibodies produced by a single B cell or B cell clone. The variableregion of each heavy chain is approximately 110 amino acids long and iscomposed of a single antibody domain.

In mammals there are only two types of light chain, which are calledlambda (λ) and kappa (κ). A light chain has two successive domains: oneconstant domain CL and one variable domain VL. The approximate length ofa light chain is 211 to 217 amino acids. Preferably the light chain is akappa (κ) light chain, and the constant domain CL is preferably C kappa(κ).

The terms “monoclonal antibody” or “monoclonal antibody composition” asused herein refer to a preparation of antibody molecules of a singleamino acid composition.

The “antibodies” according to the invention can be of any class (e.g.IgA, IgD, IgE, IgG, and IgM, preferably IgG or IgE), or subclass (e.g.,IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2, preferably IgG1), whereby bothantibodies, from which the bivalent bispecific antibody according to theinvention is derived, have an Fc part of the same subclass (e.g. IgG1,IgG4 and the like, preferably IgG1), preferably of the same allotype(e.g. Caucasian).

A “Fc part of an antibody” is a term well known to the skilled artisanand defined on the basis of papain cleavage of antibodies. Theantibodies according to the invention contain as Fc part, preferably aFc part derived from human origin and preferably all other parts of thehuman constant regions. The Fc part of an antibody is directly involvedin complement activation, C1q binding, C3 activation and Fc receptorbinding. While the influence of an antibody on the complement system isdependent on certain conditions, binding to C1q is caused by definedbinding sites in the Fc part. Such binding sites are known in the stateof the art and described e.g. by Lukas, T. J., et al., J. Immunol. 127(1981) 2555-2560; Brunhouse, R., and Cebra, J. J., Mol. Immunol. 16(1979) 907-917; Burton, D. R., et al., Nature 288 (1980) 338-344;Thommesen, J. E., et al., Mol. Immunol. 37 (2000) 995-1004; Idusogie, E.E., et al., J. Immunol. 164 (2000) 4178-4184; Hezareh, M., et al., J.Virol. 75 (2001) 12161-12168; Morgan, A., et al., Immunology 86 (1995)319-324; and EP 0 307 434. Such binding sites are e.g. L234, L235, D270,N297, E318, K320, K322, P331 and P329 (numbering according to EU indexof Kabat, see below). Antibodies of subclass IgG1, IgG2 and IgG3 usuallyshow complement activation, C1q binding and C3 activation, whereas IgG4do not activate the complement system, do not bind C1q and do notactivate C3. Preferably the Fc part is a human Fc part.

The term “chimeric antibody” refers to an antibody comprising a variableregion, i.e., binding region, from one source or species and at least aportion of a constant region derived from a different source or species,usually prepared by recombinant DNA techniques. Chimeric antibodiescomprising a murine variable region and a human constant region arepreferred. Other preferred forms of “chimeric antibodies” encompassed bythe present invention are those in which the constant region has beenmodified or changed from that of the original antibody to generate theproperties according to the invention, especially in regard to C1qbinding and/or Fc receptor (FcR) binding. Such chimeric antibodies arealso referred to as “class-switched antibodies.”. Chimeric antibodiesare the product of expressed immunoglobulin genes comprising DNAsegments encoding immunoglobulin variable regions and DNA segmentsencoding immunoglobulin constant regions. Methods for producing chimericantibodies involve conventional recombinant DNA and gene transfectiontechniques are well known in the art. See, e.g., Morrison, S. L., etal., Proc. Natl. Acad. Sci. USA 81 (1984) 6851-6855; U.S. Pat. No.5,202,238 and U.S. Pat. No. 5,204,244.

The term “humanized antibody” refers to antibodies in which theframework or “complementarity determining regions” (CDR) have beenmodified to comprise the CDR of an immunoglobulin of differentspecificity as compared to that of the parent immunoglobulin. In apreferred embodiment, a murine CDR is grafted into the framework regionof a human antibody to prepare the “humanized antibody.” See, e.g.,Riechmann, L., et al., Nature 332 (1988) 323-327; and Neuberger, M. S.,et al., Nature 314 (1985) 268-270. Particularly preferred CDRscorrespond to those representing sequences recognizing the antigensnoted above for chimeric antibodies. Other forms of “humanizedantibodies” encompassed by the present invention are those in which theconstant region has been additionally modified or changed from that ofthe original antibody to generate the properties according to theinvention, especially in regard to C1q binding and/or Fc receptor (FcR)binding.

The term “human antibody”, as used herein, is intended to includeantibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies are well-known in thestate of the art (van Dijk, M. A., and van de Winkel, J. G., Curr. Opin.Chem. Biol. 5 (2001) 368-374). Human antibodies can also be produced intransgenic animals (e.g., mice) that are capable, upon immunization, ofproducing a full repertoire or a selection of human antibodies in theabsence of endogenous immunoglobulin production. Transfer of the humangerm-line immunoglobulin gene array in such germ-line mutant mice willresult in the production of human antibodies upon antigen challenge(see, e.g., Jakobovits, A., et al., Proc. Natl. Acad. Sci. USA 90 (1993)2551-2555; Jakobovits, A., et al., Nature 362 (1993) 255-258;Brüggemann, M., et al., Year Immunol. 7 (1993) 33-40). Human antibodiescan also be produced in phage display libraries (Hoogenboom, H. R., andWinter, G., J. Mol. Biol. 227 (1992) 381-388; Marks, J. D., et al., J.Mol. Biol. 222 (1991) 581-597). The techniques of Cole, S. P. C., et al.and Boerner et al. are also available for the preparation of humanmonoclonal antibodies (Cole, S. P. C., et al., Monoclonal Antibodies andCancer Therapy, Alan R. Liss, Inc., New York (1986), pp. 77-96; andBoerner, P., et al., J. Immunol. 147 (1991) 86-95). As already mentionedfor chimeric and humanized antibodies according to the invention theterm “human antibody” as used herein also comprises such antibodieswhich are modified in the constant region to generate the propertiesaccording to the invention, especially in regard to C1q binding and/orFcR binding, e.g. by “class switching” i.e. change or mutation of Fcparts (e.g. from IgG1 to IgG4 and/or IgG1/IgG4 mutation).

The term “recombinant human antibody”, as used herein, is intended toinclude all human antibodies that are prepared, expressed, created orisolated by recombinant means, such as antibodies isolated from a hostcell such as a NS0 or CHO cell or from an animal (e.g. a mouse) that istransgenic for human immunoglobulin genes or antibodies expressed usinga recombinant expression vector transfected into a host cell. Suchrecombinant human antibodies have variable and constant regions in arearranged form. The recombinant human antibodies according to theinvention have been subjected to in vivo somatic hypermutation. Thus,the amino acid sequences of the VH and VL regions of the recombinantantibodies are sequences that, while derived from and related to humangerm line VH and VL sequences, may not naturally exist within the humanantibody germ line repertoire in vivo.

The “variable domain” (variable domain of a light chain (VL), variableregion of a heavy chain (VH)) as used herein denotes each of the pair oflight and heavy chains which is involved directly in binding theantibody to the antigen. The domains of variable human light and heavychains have the same general structure and each domain comprises fourframework (FR) regions whose sequences are widely conserved, connectedby three “hypervariable regions” (or complementarity determiningregions, CDRs). The framework regions adopt a β-sheet conformation andthe CDRs may form loops connecting the β-sheet structure. The CDRs ineach chain are held in their three-dimensional structure by theframework regions and form together with the CDRs from the other chainthe antigen binding site. The antibody heavy and light chain CDR3regions play a particularly important role in the bindingspecificity/affinity of the antibodies according to the invention andtherefore provide a further object of the invention.

The terms “hypervariable region” or “antigen-binding portion of anantibody” when used herein refer to the amino acid residues of anantibody which are responsible for antigen-binding. The hypervariableregion comprises amino acid residues from the “complementaritydetermining regions” or “CDRs”. “Framework” or “FR” regions are thosevariable domain regions other than the hypervariable region residues asherein defined. Therefore, the light and heavy chains of an antibodycomprise from N- to C-terminus the domains FR1, CDR1, FR2, CDR2, FR3,CDR3, and FR4. CDRs on each chain are separated by such framework aminoacids. Especially, CDR3 of the heavy chain is the region whichcontributes most to antigen binding. CDR and FR regions are determinedaccording to the standard definition of Kabat et al., Sequences ofProteins of Immunological Interest, 5th ed., Public Health Service,National Institutes of Health, Bethesda, Md. (1991).

The “constant domains” of the heavy chain and of the light chain are notinvolved directly in binding of an antibody to an antigen, but exhibitvarious effector functions. Depending on the amino acid sequence of theconstant region of their heavy chains, antibodies or immunoglobulins aredivided into the classes:

The term “bivalent, bispecific antibody” as used herein refers to anantibody as described above in which each of the two pairs of heavychain and light chain (HC/LC) is specifically binding to a differentantigen, i.e. the first heavy and the first light chain (originatingfrom an antibody against a first antigen) are specifically bindingtogether to a first antigen, and, the second heavy and the second lightchain (originating from an antibody against a second antigen) arespecifically binding together to a second antigen (as depicted in FIG.2); such bivalent, bispecific antibodies are capable of specificallybinding to two different antigens at the same time, and not to more thantwo antigens, in contrary to, on the one hand a monospecific antibodycapable of binding only to one antigen, and on the other hand e.g. atetravalent, tetraspecific antibody which can bind to four antigenmolecules at the same time.

According to the invention, the ratio of a desired bivalent, bispecificantibody compared to undesired side products can be improved by thereplacement of certain domains in only one pair of heavy chain and lightchain (HC/LC). While the first of the two HC/LC pairs originates from anantibody specifically binding to a first antigen and is left essentiallyunchanged, the second of the two HC/LC pairs originates from an antibodyspecifically binding to a second antigen, and is altered by thefollowing replacement:

light chain: replacement of the variable light chain domain VL by thevariable heavy chain domain VH of said antibody specifically binding toa second antigen, and the constant light chain domain CL by the constantheavy chain domain CH1 of said antibody specifically binding to a secondantigen, and

heavy chain: replacement of the variable heavy chain domain VH by thevariable light chain domain VL of said antibody specifically binding toa second antigen, and the constant heavy chain domain CH1 by theconstant light chain domain CL of said antibody specifically binding toa second antigen.

Thus the resulting bivalent, bispecific antibodies are artificialantibodies which comprise

a) the light chain and heavy chain of an antibody specifically bindingto a first antigen; and

b) the light chain and heavy chain of an antibody specifically bindingto a second antigen, wherein said light chain (of an antibodyspecifically binding to a second antigen) contains a variable domain VHinstead of VL

and a constant domain CH1 instead of CL

wherein said heavy chain (of an antibody specifically binding to asecond antigen) contains a variable domain VL instead of VH

and a constant domain CL instead of CH1.

In an additional aspect of the invention such improved ratio of adesired bivalent, bispecific antibody compared to undesired sideproducts can be further improved by one of the following twoalternatives:

A) First Alternative (See FIG. 3):

The CH3 domains of said bivalent, bispecific antibody according to theinvention can be altered by the “knob-into-holes” technology which isdescribed in detail with several examples in e.g. WO 96/027011, RidgwayJ. B., et al., Protein Eng 9 (1996) 617-621; and Merchant, A. M., etal., Nat Biotechnol 16 (1998) 677-681. In this method the interactionsurfaces of the two CH3 domains are altered to increase theheterodimerisation of both heavy chains containing these two CH3domains. Each of the two CH3 domains (of the two heavy chains) can bethe “knob”, while the other is the “hole”. The introduction of adisulfide bridge stabilizes the heterodimers (Merchant, A. M, et al.,Nature Biotech 16 (1998) 677-681; Atwell, S., et al. J. Mol. Biol. 270(1997) 26-35) and increases the yield.

Therefore in preferred embodiment the CH3 domains of a bivalent,bispecific antibody wherein the first CH3 domain and second CH3 domaineach meet at an interface which comprises an original interface betweenthe antibody CH3 domains are altered by the “knob-into-holes” technologyincluding further stabilization by introduction of a disulfide bridge inthe CH3 domains (described in WO 96/027011, Ridgway, J. B., et al.,Protein Eng 9 (1996) 617-621; Merchant, A. M., et al, Nature Biotech 16(1998) 677-681; and Atwell, S., et al., J. Mol. Biol. 270 (1997) 26-35)to promote the formation of the bivalent, bispecific antibody.

Thus in one aspect of the invention said bivalent, bispecific antibodyis characterized in that the CH3 domain of one heavy chain and the CH3domain of the other heavy chain each meet at an interface whichcomprises an original interface between the antibody CH3 domains;

wherein said interface is altered to promote the formation of thebivalent, bispecific antibody, wherein the alteration is characterizedin that:

a) the CH3 domain of one heavy chain is altered,

so that within the original interface the CH3 domain of one heavy chainthat meets the original interface of the CH3 domain of the other heavychain within the bivalent, bispecific antibody, an amino acid residue isreplaced with an amino acid residue having a larger side chain volume,thereby generating a protuberance within the interface of the CH3 domainof one heavy chain which is positionable in a cavity within theinterface of the CH3 domain of the other heavy chain andb) the CH3 domain of the other heavy chain is altered,so that within the original interface of the second CH3 domain thatmeets the original interface of the first CH3 domain within thebivalent, bispecific antibodyan amino acid residue is replaced with an amino acid residue having asmaller side chain volume, thereby generating a cavity within theinterface of the second CH3 domain within which a protuberance withinthe interface of the first CH3 domain is positionable.

Preferably said amino acid residue having a larger side chain volume isselected from the group consisting of arginine (R), phenylalanine (F),tyrosine (Y), tryptophan (W).

Preferably said amino acid residue having a smaller side chain volume isselected from the group consisting of alanine (A), serine (S), threonine(T), valine (V).

In one aspect of the invention both CH3 domains are further altered theintroduction of cysteine (C) as amino acid in the correspondingpositions of each CH3 domain such that a disulfide bridge between bothCH3 domains can be formed.

In another preferred embodiment of the invention both CH3 domains arealtered by the use of residues R409D; K370E (K409D) for knobs residuesand D399K; E357K for hole residues described eg. in EP 1870459A1; or

B) Second Alternative (See FIG. 4):

by the replacement of one constant heavy chain domain CH3 by a constantheavy chain domain CH1; and the other constant heavy chain domain CH3 isreplaced by a constant light chain domain CL.

The constant heavy chain domain CH1 by which the heavy chain domain CH3is replaced can be of any Ig class (e.g. IgA, IgD, IgE, IgG, and IgM),or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2).

The constant light chain domain CL by which the heavy chain domain CH3is replaced can be of the lambda (λ) or kappa (κ) type, preferably thekappa (κ) type.

Thus one preferred embodiment of the invention is a bivalent, bispecificantibody, comprising:

a) the light chain and heavy chain of an antibody specifically bindingto a first antigen; and

b) the light chain and heavy chain of an antibody specifically bindingto a second antigen,

wherein the variable domains VL and VH are replaced by each other,

and

wherein the constant domains CL and CH1 are replaced by each other,

and wherein optionally

c) the CH3 domain of one heavy chain and the CH3 domain of the otherheavy chain each meet at an interface which comprises an originalinterface between the antibody CH3 domains;

wherein said interface is altered to promote the formation of thebivalent, bispecific antibody, wherein the alteration is characterizedin that:

ca) the CH3 domain of one heavy chain is altered,

so that within the original interface the CH3 domain of one heavy chainthat meets the original interface of the CH3 domain of the other heavychain within the bivalent, bispecific antibody, an amino acid residue isreplaced with an amino acid residue having a larger side chain volume,thereby generating a protuberance within the interface of the CH3 domainof one heavy chain which is positionable in a cavity within theinterface of the CH3 domain of the other heavy chain andcb) the CH3 domain of the other heavy chain is altered,so that within the original interface of the second CH3 domain thatmeets the original interface of the first CH3 domain within thebivalent, bispecific antibodyan amino acid residue is replaced with an amino acid residue having asmaller side chain volume, thereby generating a cavity within theinterface of the second CH3 domain within which a protuberance withinthe interface of the first CH3 domain is positionable;ord) one constant heavy chain domain CH3 is replaced by a constant heavychain domain CH1; and the other constant heavy chain domain CH3 isreplaced by a constant light chain domain CL

The terms “antigen” or “antigen molecule” as used herein are usedinterchangeable and refer to all molecules that can be specificallybound by an antibody. The bivalent, bispecific antibody is specificallybinding to a first antigen and a second distinct antigen. The term“antigens” as used herein include e.g. proteins, different epitopes onproteins (as different antigens within the meaning of the invention),and polysaccharides. This mainly includes parts (coats, capsules, cellwalls, flagella, fimbrae, and toxins) of bacteria, viruses, and othermicroorganisms. Lipids and nucleic acids are antigenic only whencombined with proteins and polysaccharides. Non-microbial exogenous(non-self) antigens can include pollen, egg white, and proteins fromtransplanted tissues and organs or on the surface of transfused bloodcells. Preferably the antigen is selected from the group consisting ofcytokines, cell surface proteins, enzymes and receptors cytokines, cellsurface proteins, enzymes and receptors.

Tumor antigens are those antigens that are presented by MHC I or MHC IImolecules on the surface of tumor cells. These antigens can sometimes bepresented by tumor cells and never by the normal ones. In this case,they are called tumor-specific antigens (TSAs) and typically result froma tumor specific mutation. More common are antigens that are presentedby tumor cells and normal cells, and they are called tumor-associatedantigens (TAAs). Cytotoxic T lymphocytes that recognized these antigensmay be able to destroy the tumor cells before they proliferate ormetastasize. Tumor antigens can also be on the surface of the tumor inthe form of, for example, a mutated receptor, in which case they will berecognized by B cells.

In one preferred embodiment at least one of the two different antigens(first and second antigen), to which the bivalent, bispecific antibodyspecifically binds to, is a tumor antigen.

In another preferred embodiment both of the two different antigens(first and second antigen), to which the bivalent, bispecific antibodyspecifically binds to, are tumor antigens; in this case the first andsecond antigen can also be two different epitopes at the same tumorspecific protein.

In another preferred embodiment one of the two different antigens (firstand second antigen), to which the bivalent, bispecific antibodyspecifically binds to, is a tumor antigen and the other is an effectorcell antigen, as e.g. an T-Cell receptor, CD3, CD16 and the like.

In another preferred embodiment one of the two different antigens (firstand second antigen), to which the bivalent, bispecific antibodyspecifically binds to, is a tumor antigen and the other is ananti-cancer substance such as a toxin or a kinase inhibitor.

As used herein, “specifically binding” or “binds specifically to” refersto an antibody specifically binding an antigen. Preferably the bindingaffinity of the antibody specifically binding this antigen is ofKD-value of 10⁻⁹ mol/l or lower (e.g. 10⁻¹⁰ mol/l), preferably with aKD-value of 10⁻¹⁰ mol/l or lower (e.g. 10⁻¹² mol/l). The bindingaffinity is determined with a standard binding assay, such as surfaceplasmon resonance technique (Biacore®).

The term “epitope” includes any polypeptide determinant capable ofspecific binding to an antibody. In certain embodiments, epitopedeterminant include chemically active surface groupings of moleculessuch as amino acids, sugar side chains, phosphoryl, or sulfonyl, and, incertain embodiments, may have specific three dimensional structuralcharacteristics, and or specific charge characteristics. An epitope is aregion of an antigen that is bound by an antibody. In certainembodiments, an antibody is said to specifically bind an antigen when itpreferentially recognizes its target antigen in a complex mixture ofproteins and/or macromolecules.

An further embodiment of the invention is a method for the preparationof a bivalent, bispecific antibody according to the invention comprising

a) transforming a host cell with

vectors comprising nucleic acid molecules encoding the light chain andheavy chain of an antibody specifically binding to a first antigen, and

vectors comprising nucleic acid molecules encoding the light chain andheavy chain of an antibody specifically binding to a second antigen,

wherein the variable domains VL and VH are replaced by each other,

and,

wherein the constant domains CL and CH1 are replaced by each other;

b) culturing the host cell under conditions that allow synthesis of saidantibody molecule; and

c) recovering said antibody molecule from said culture.

In general there are two vectors encoding the light chain and heavychain of said antibody specifically binding to a first antigen, andfurther two vectors encoding the light chain and heavy chain of saidantibody specifically binding to a second antigen. One of the twovectors is encoding the respective light chain and the other of the twovectors is encoding the respective heavy chain. However in analternative method for the preparation of a bivalent, bispecificantibody according to the invention, only one first vector encoding thelight chain and heavy chain of the antibody specifically binding to afirst antigen and only one second vector encoding the light chain andheavy chain of the antibody specifically binding to a second antigen canbe used for transforming the host cell.

The invention encompasses a method for the preparation of the antibodiescomprising culturing the corresponding host cells under conditions thatallow synthesis of said antibody molecules and recovering saidantibodies from said culture, e.g. by expressing

a first nucleic acid sequence encoding the light chain of an antibodyspecifically binding to a first antigen;

a second nucleic acid sequence encoding the heavy chain of said antibodyspecifically binding to a first antigen;

a third nucleic acid sequence encoding the light chain of an antibodyspecifically binding to a second antigen, wherein the variable lightchain domain VL is replaced by the variable heavy chain domain VH, andwherein the constant light chain domain CL is replaced by the constantheavy chain domain CH1; and

a fourth nucleic acid sequence encoding the heavy chain of said antibodyspecifically binding to a second antigen, wherein the variable heavychain domain VH is replaced by the variable light chain domain VL, andwherein the constant heavy chain domain CH1 is replaced by the constantlight chain domain CL.

A further embodiment of the invention is a host cell comprising

vectors comprising nucleic acid molecules encoding the light chain andheavy chain of an antibody specifically binding to a first antigen, and

vectors comprising nucleic acid molecules encoding the light chain andheavy chain of an antibody specifically binding to a second antigen,

wherein the variable domains VL and VH are replaced by each other,

and

wherein the constant domains CL and CH1 are replaced by each other.

A further embodiment of the invention is a host cell comprising

a) a vector comprising a nucleic acid molecule encoding the light chainand a vector comprising a nucleic acid molecule encoding the heavychain, of an antibody specifically binding to a first antigen, and

b) a vector comprising a nucleic acid molecule encoding the light chainand a vector comprising a nucleic acid molecule encoding the heavychain, of an antibody specifically binding to a second antigen,

wherein the variable domains VL and VH are replaced by each other,

and

wherein the constant domains CL and CH1 are replaced by each other.

A further embodiment of the invention is a composition, preferably apharmaceutical or a diagnostic composition of the bivalent, bispecificantibody according to the invention.

A further embodiment of the invention is a pharmaceutical compositioncomprising a bivalent, bispecific antibody according to the inventionand at least one pharmaceutically acceptable excipient.

A further embodiment of the invention is a method for the treatment of apatient in need of therapy, characterized by administering to thepatient a therapeutically effective amount of a bivalent, bispecificantibody according to the invention.

The term “nucleic acid or nucleic acid molecule”, as used herein, isintended to include DNA molecules and RNA molecules. A nucleic acidmolecule may be single-stranded or double-stranded, but preferably isdouble-stranded DNA.

As used herein, the expressions “cell,” “cell line,” and “cell culture”are used interchangeably and all such designations include progeny.Thus, the words “transformants” and “transformed cells” include theprimary subject cell and cultures derived therefrom without regard forthe number of transfers. It is also understood that all progeny may notbe precisely identical in DNA content, due to deliberate or inadvertentmutations. Variant progeny that have the same function or biologicalactivity as screened for in the originally transformed cell areincluded. Where distinct designations are intended, it will be clearfrom the context.

The term “transformation” as used herein refers to process of transferof a vectors/nucleic acid into a host cell. If cells without formidablecell wall barriers are used as host cells, transfection is carried oute.g. by the calcium phosphate precipitation method as described byGraham, F. L., and van der Eb, A. J., Virology 52 (1973) 456-467.However, other methods for introducing DNA into cells such as byelectroporation, nucleofection, nuclear injection or by protoplastfusion may also be used. If prokaryotic cells or cells which containsubstantial cell wall constructions are used, e.g. one method oftransfection is calcium treatment using calcium chloride as described byCohen, S. N, et al, PNAS. 69 (1972) 2110-2114.

Recombinant production of antibodies using transformation is well-knownin the state of the art and described, for example, in the reviewarticles of Makrides, S. C., Protein Expr. Purif. 17 (1999) 183-202;Geisse, S., et al., Protein Expr. Purif. 8 (1996) 271-282; Kaufman, R.J., Mol. Biotechnol. 16 (2000) 151-160; Werner, R. G., et al.,Arzneimittelforschung 48 (1998) 870-880 as well as in U.S. Pat. No.6,331,415 and U.S. Pat. No. 4,816,567.

As used herein, “expression” refers to the process by which a nucleicacid is transcribed into mRNA and/or to the process by which thetranscribed mRNA (also referred to as transcript) is subsequently beingtranslated into peptides, polypeptides, or proteins. The transcripts andthe encoded polypeptides are collectively referred to as gene product.If the polynucleotide is derived from genomic DNA, expression in aeukaryotic cell may include splicing of the mRNA.

A “vector” is a nucleic acid molecule, in particular self-replicating,which transfers an inserted nucleic acid molecule into and/or betweenhost cells. The term includes vectors that function primarily forinsertion of DNA or RNA into a cell (e.g., chromosomal integration),replication of vectors that function primarily for the replication ofDNA or RNA, and expression vectors that function for transcriptionand/or translation of the DNA or RNA. Also included are vectors thatprovide more than one of the functions as described.

An “expression vector” is a polynucleotide which, when introduced intoan appropriate host cell, can be transcribed and translated into apolypeptide. An “expression system” usually refers to a suitable hostcell comprised of an expression vector that can function to yield adesired expression product.

The bivalent, bispecific antibodies according to the invention arepreferably produced by recombinant means. Such methods are widely knownin the state of the art and comprise protein expression in prokaryoticand eukaryotic cells with subsequent isolation of the antibodypolypeptide and usually purification to a pharmaceutically acceptablepurity. For the protein expression, nucleic acids encoding light andheavy chains or fragments thereof are inserted into expression vectorsby standard methods. Expression is performed in appropriate prokaryoticor eukaryotic host cells like CHO cells, NS0 cells, SP2/0 cells, HEK293cells, COS cells, PER.C6 cells, yeast, or E. coli cells, and theantibody is recovered from the cells (supernatant or cells after lysis).The bivalent, bispecific antibodies may be present in whole cells, in acell lysate, or in a partially purified or substantially pure form.Purification is performed in order to eliminate other cellularcomponents or other contaminants, e.g. other cellular nucleic acids orproteins, by standard techniques, including alkaline/SDS treatment,column chromatography and others well known in the art. See Ausubel, F.,et al., ed., Current Protocols in Molecular Biology, Greene Publishingand Wiley Interscience, New York (1987).

Expression in NS0 cells is described by, e.g., Barnes, L. M., et al.,Cytotechnology 32 (2000) 109-123; and Barnes, L. M., et al., Biotech.Bioeng. 73 (2001) 261-270. Transient expression is described by, e.g.,Durocher, Y., et al., Nucl. Acids. Res. 30 (2002) E9. Cloning ofvariable domains is described by Orlandi, R., et al., Proc. Natl. Acad.Sci. USA 86 (1989) 3833-3837; Carter, P., et al., Proc. Natl. Acad. Sci.USA 89 (1992) 4285-4289; and Norderhaug, L., et al., J. Immunol. Methods204 (1997) 77-87. A preferred transient expression system (HEK 293) isdescribed by Schlaeger, E.-J., and Christensen, K., in Cytotechnology 30(1999) 71-83 and by Schlaeger, E.-J., in J. Immunol. Methods 194 (1996)191-199.

The control sequences that are suitable for prokaryotes, for example,include a promoter, optionally an operator sequence, and a ribosomebinding site. Eukaryotic cells are known to utilize promoters, enhancersand polyadenylation signals.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading frame. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

The bivalent, bispecific antibodies are suitably separated from theculture medium by conventional immunoglobulin purification proceduressuch as, for example, protein A-Sepharose, hydroxylapatitechromatography, gel electrophoresis, dialysis, or affinitychromatography. DNA and RNA encoding the monoclonal antibodies arereadily isolated and sequenced using conventional procedures. Thehybridoma cells can serve as a source of such DNA and RNA. Onceisolated, the DNA may be inserted into expression vectors, which arethen transfected into host cells such as HEK 293 cells, CHO cells, ormyeloma cells that do not otherwise produce immunoglobulin protein, toobtain the synthesis of recombinant monoclonal antibodies in the hostcells.

Amino acid sequence variants (or mutants) of the bivalent, bispecificantibody are prepared by introducing appropriate nucleotide changes intothe antibody DNA, or by nucleotide synthesis. Such modifications can beperformed, however, only in a very limited range, e.g. as describedabove. For example, the modifications do not alter the above mentionedantibody characteristics such as the IgG isotype and antigen binding,but may improve the yield of the recombinant production, proteinstability or facilitate the purification.

The following examples, sequence listing and figures are provided to aidthe understanding of the present invention, the true scope of which isset forth in the appended claims. It is understood that modificationscan be made in the procedures set forth without departing from thespirit of the invention.

Sequence Listing

SEQ ID NO: 1 amino acid sequence of wild type <IGF-1R> antibody heavychain

SEQ ID NO: 2 amino acid sequence of wild type <IGF-1R> antibody lightchain

SEQ ID NO: 3 amino acid sequence of the heavy chain* (HC*) of <IGF-1R>VL-VH/CL-CH1 exchange antibody, wherein the heavy chain domain VH isreplaced by the light chain domain VL, and the heavy chain domain CH1 isreplaced by the light chain domain CL.

SEQ ID NO: 4 amino acid sequence of the light chain* (LC*) of <IGF-1R>VL-VH/CL-CH1 exchange antibody, wherein the light chain domain VL isreplaced by the heavy chain domain VH, and the light chain domain CL isreplaced by the heavy chain domain CH1.

SEQ ID NO: 5 amino acid sequence of IGF-1R ectodomain His-Streptavidinbinding peptide-tag (IGF-1R-His-SBP ECD)

SEQ ID NO: 6 amino acid sequence of wild type Angiopoietin-2<ANGPT2>antibody heavy chain

SEQ ID NO: 7 amino acid sequence of wild type Angiopoietin-2<ANGPT2>antibody light chain

SEQ ID NO: 8 amino acid sequence of the heavy chain* (HC*) of <ANGPT2>VL-VH/CL-CH1 exchange antibody, wherein the heavy chain domain VH isreplaced by the light chain domain VL, and the heavy chain domain CH1 isreplaced by the light chain domain CL.

SEQ ID NO: 9 amino acid sequence of the light chain* (LC*) of <ANGPT2>VL-VH/CL-CH1 exchange antibody, wherein the light chain domain VL isreplaced by the heavy chain domain VH, and the light chain domain CL isreplaced by the heavy chain domain CH1.

SEQ ID NO: 10 amino acid sequence of CH3 domain (Knobs) with a T366Wexchange for use in the knobs-into-holes technology

SEQ ID NO: 11 amino acid sequence CH3 domain (Hole) with a T366S, L368A,Y407V exchange for use in the knobs-into-holes technology

SEQ ID NO: 12 amino acid sequence of wild type <VEGF> antibody heavychain

SEQ ID NO: 13 and 14 amino acid sequence of wild type <VEGF> antibodylight chain with and without leader

SEQ ID NO: 15 amino acid sequence of the heavy chain* (HC*) of <ANGPT2>VL-VH/CL-CH1 exchange antibody, wherein the heavy chain domain VH isreplaced by the light chain domain VL, and the heavy chain domain CH1 isreplaced by the light chain domain CL and the CH3 domain carries anamino acid sequence with a T366S, L368A, Y407V exchange (Hole) for usein the knobs-into-holes technology

SEQ ID NO: 16 amino acid sequence of wild type <VEGF> antibody heavychain, wherein the CH3 domain carries an amino acid sequence with aT366W (Knobs) exchange for use in the knobs-into-holes technology

SEQ ID NO: 17 amino acid sequence of the heavy chain(G)* (HC*) of<ANGPT2> VL-VH/CL-CH1 exchange antibody, wherein the heavy chain domainVH is replaced by the light chain domain VL, and the heavy chain domainCH1 is replaced by the light chain domain CL with an additional Glycininsertion.

SEQ ID NO: 18 amino acid sequence of the light chain(G)* (LC*) of<ANGPT2> VL-VH/CL-CH1 exchange antibody, wherein the light chain domainVL is replaced by the heavy chain domain VH, and the light chain domainCL is replaced by the heavy chain domain CH1 with an additional Glycininsertion.

DESCRIPTION OF THE FIGURES

FIG. 1 Schematic figure of IgG, a naturally occurring whole antibodyspecific for one antigen with two pairs of heavy and light chain whichcomprise variable and constant domains in a typical order.

FIG. 2 Schematic figure of a bivalent, bispecific antibody, comprising:a) the light chain and heavy chain of an antibody specifically bindingto a first antigen; and b) the light chain and heavy chain of anantibody specifically binding to a second antigen, wherein the variabledomains VL and VH are replaced by each other, and wherein the constantdomains CL and CH1 are replaced by each other.

FIG. 3 Schematic figure of a bivalent, bispecific antibody, comprising:a) the light chain and heavy chain of an antibody specifically bindingto a first antigen; and b) the light chain and heavy chain of anantibody specifically binding to a second antigen, wherein the variabledomains VL and VH are replaced by each other, and wherein the constantdomains CL and CH1 are replaced by each other, and wherein the CH3domains of both heavy chains are altered by the knobs-into-holestechnology.

FIG. 4 Schematic figure of a bivalent, bispecific antibody, comprising:a) the light chain and heavy chain of an antibody specifically bindingto a first antigen; and b) the light chain and heavy chain of anantibody specifically binding to a second antigen, wherein the variabledomains VL and VH are replaced by each other, and wherein the constantdomains CL and CH1 are replaced by each other, and wherein one of theconstant heavy chain domains CH3 of both heavy chains is replaced by aconstant heavy chain domain CH1, and the other constant heavy chaindomain CH3 is replaced by a constant light chain domain CL.

FIG. 5 Protein sequence scheme of the heavy chain* <IGF-1R> HC* of the<IGF-1R> VL-VH/CL-CH1 exchange antibody

FIG. 6 Protein sequence scheme of the light chain* <IGF-1R> LC* of the<IGF-1R> VL-VH/CL-CH1 exchange antibody

FIG. 7 Plasmid map of heavy chain* <IGF-1R> HC* expression vectorpUC-HC*-IGF-1R

FIG. 8 Plasmid map of light chain* <IGF-1R> LC* expression vectorpUC-LC*-IGF-1R

FIG. 9 Plasmid map of the 4700-Hyg-OriP expression vector

FIG. 10 SDS-PAGE of monospecific, bivalent <IGF-1R> VL-VH/CL-CH1exchange antibody (IgG1*) with HC* and LC* isolated byimmunoprecipitation with Protein A Agarose from cell culturesupernatants after transient transfection of HEK293E cells.

FIG. 11 Binding of monospecific <IGF-1R> VL-VH/CL-CH1 exchange antibodyand wildtype <IGF-1R> antibody to the IGF-1R ECD in an ELISA-basedbinding assay.

FIG. 12 Plasmid map of heavy chain* <ANGPT2> HC* expression vectorpUC-HC*-ANGPT2>

FIG. 13 Plasmid map of light chain* <ANGPT2> LC* expression vectorpUC-LC*-ANGPT2>

FIG. 14 Reduced and non-reduced SDS-PAGE from purification of mix of A)monospecific <IGF-1R> VL-VH/CL-CH1 exchange antibody, B) bispecific<ANGPT2-IGF-1R> VL-VH/CL-CH1 exchange antibody and C)<ANGPT2> wildtypeantibodies (“Bispecific VL-VH/CL-CH1 exchange mix”) from cell culturesupernatants by Protein A affinity chromatography followed by sizeexclusion chromatography and concentration

FIG. 15 Assay principle of cellular FACS IGF-1R-ANGPT2 bridging assay on124 IGF-1R expressing cells to detect the presence of functionalbispecific <ANGPT2-IGF-1R> VL-VH/CL-CH1 exchange antibody

FIG. 16 Results for Samples A to G of cellular FACS IGF-1R-ANGPT2bridging assay on 124 IGF-1R expressing cells to detect the presence offunctional bispecific <ANGPT2-IGF-1R> VL-VH/CL-CH1 exchange antibody incell culture supernatants.

Cell Sample IGF-1R Antibody hANGPT2 Detection antibody Detection A I24untreated — <Ang-2>mIgG1-Biotin SA-PE B I24 untreated 2 μg/mL hANGPT2<Ang-2>mIgG1-Biotin SA-PE C I24 bispecific <ANGPT2- 2 μg/mL hANGPT2<Ang-2>mIgG1-Biotin SA-PE IGF-1R> VL-VH/CL- CH1 exchange antibody mix DI24 Wildtype mix 2 μg/mL hANGPT2 <Ang-2>mIgG1-Biotin SA-PE E I24<ANGPT2> wildtype 2 μg/mL hANGPT2 <Ang-2>mIgG1-Biotin SA-PE antibody FI24 <IGF-1R> wildtype 2 μg/mL hANGPT2 <Ang-2>mIgG1-Biotin SA-PE antibodyG I24 monospecific <IGF- 2 μg/mL hANGPT2 <Ang-2>mIgG1-Biotin SA-PE1R>VL-VH/CL-CH1 exchange antibody

FIG. 17 Results for Samples A to G of cellular FACS IGF-1R-ANGPT2bridging assay on I24 IGF-1R expressing cells to detect the presence offunctional bispecific <ANGPT2-IGF-1R> VL-VH/CL-CH1 exchange antibody

Cell Sample IGF-1R Antibody hANGPT2 Detection antibody Detection A I24untreated — <Ang-2>mIgG1-Biotin SA-PE B I24 untreated 2 μg/mL hANGPT2<Ang-2>mIgG1-Biotin SA-PE C I24 Bispecific VL- 2 μg/mL hANGPT2mIgG1-Biotin-Isotype control SA-PE VH/CL-CH1 exchange mix D I24Bispecific VL- 2 μg/mL hANGPT2 <Ang-2>mIgG1-Biotin SA-PE VH/CL-CH1exchange mix E I24 Wildtype mix 2 μg/mL hANGPT2 <Ang-2>mIgG1-BiotinSA-PE F I24 <ANGPT2> wildtype 2 μg/mL hANGPT2 <Ang-2>mIgG1-Biotin SA-PEantibody G I24 <IGF-1R> wildtype 2 μg/mL hANGPT2 <Ang-2>mIgG1-BiotinSA-PE antibody

FIG. 18 Scheme of the IGF-1R ECD binding ELISA

FIG. 19 Scheme of the ANGPT2 binding ELISA

FIG. 20 Scheme of the VEGF-ANGPT2 bridging ELISA

FIG. 21 Scheme of the ANGPT2-VEGF bridging Biacore assay. PentaHis tagdisclosed as SEQ ID NO: 19.

FIG. 22 A) SDS-PAGE of the purification of <IGF-1R> VL-VH/CL-CH1exchange antibody, purified <IGF-1R> VL-VH/CL-CH1 exchange antibodycorresponds to SEC pool concentrated; B) size exclusion chromatographyof the purified <IGF-1 R>VL-VH/CL-CH1 exchange antibody

FIG. 23 A) SDS-PAGE of the purification of <ANGPT2> VL-VH/CL-CH1exchange antibody, purified <ANPT2> VL-VH/CL-CH1 exchange antibodycorresponds to SEC pool concentrated); B) size exclusion chromatographyof the purified <ANGPT2> VL-VH/CL-CH1 exchange antibody

FIG. 24 Binding of monospecific <ANGPt2> VL-VH/CL-CH1 exchange antibodyand wildtype <ANGPt2> antibody to ANGPT2 in an ELISA-based bindingassay.

FIG. 25 Biacore analysis of binding of monospecific <ANGPt2>VL-VH/CL-CH1 exchange antibody and wildtype <ANGPt2> antibody to ANGPT2

FIG. 26 SDS-PAGE reduced and non-reduced of the elution fractions fromsize exclusion chromatography of the bispecific <VEGF-ANGPT2>VL-VH/CL-CH1 exchange antibody

FIG. 27 Assignment of bands in SDS-PAGE by mass spectrometry of nativefractions. The position of proteins identified by mass spectrometry inthe respective unreduced SDS-PAGE is indicated

FIG. 28 Analysis of the elution fractions 5 and 9 from size exclusionchromatography of the bispecific <VEGF-ANGPT2> VL-VH/CL-CH1 exchangeantibody in the VEGF-ANGPT2 bridging ELISA. A bispecific and tetravalentantibody TvG6-Ang23 recognizing ANGPT2 and VEGF simultaneously isincluded as positive control.

FIG. 29 Surface Plasmon resonance analysis of the elution fractions 5and 9 from size exclusion chromatography of the bispecific <VEGF-ANGPT2>VL-VH/CL-CH1 exchange antibody in the VEGF-ANGPT2 bridging Biacoreassay. A bispecific and tetravalent antibody TvG6-Ang23 recognizingANGPT2 and VEGF simultaneously is included as positive control. A)negative control; B) Fraction 5; C) Positive control; D) Fraction 9.

FIG. 30 Scheme of the bispecific <VEGF-ANGPT2> VL-VH/CL-CH1 exchangeantibody with knobs-in-holes for heterodimerization

FIG. 31 Biacore analysis of binding of monospecific <ANGPT2>VL-VH(G)/CL-CH1(G) exchange antibody and wildtype <ANGPT2> antibody toANGPT2. A) Wildtype Biacore first KD determination; B) Wildtype Biacoresecond KD determination; C) Exchange antibody first KD determination; D)Exchange antibody second KD determination.

EXAMPLES

Materials & General Methods

General information regarding the nucleotide sequences of humanimmunoglobulins light and heavy chains is given in: Kabat, E. A., etal., Sequences of Proteins of Immunological Interest, 5th ed., PublicHealth Service, National Institutes of Health, Bethesda, Md. (1991).Amino acids of antibody chains are numbered and referred to according toEU numbering (Edelman, G. M., et al., Proc. Natl. Acad. Sci. USA 63(1969) 78-85; Kabat, E. A., et al., Sequences of Proteins ofImmunological Interest, 5th ed., Public Health Service, NationalInstitutes of Health, Bethesda, Md., (1991)).

Recombinant DNA Techniques

Standard methods were used to manipulate DNA as described in Sambrook,J. et al., Molecular cloning: A laboratory manual; Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989. The molecularbiological reagents were used according to the manufacturer'sinstructions.

Gene Synthesis

Desired gene segments were prepared from oligonucleotides made bychemical synthesis. The 600-1800 bp long gene segments, which areflanked by singular restriction endonuclease cleavage sites, wereassembled by annealing and ligation of oligonucleotides including PCRamplification and subsequently cloned via the indicated restrictionsites e.g. KpnI/SacI or AscI/PacI into a pPCRScript (Stratagene) basedpGA4 cloning vector. The DNA sequences of the subcloned gene fragmentswere confirmed by DNA sequencing. Gene synthesis fragments were orderedaccording to given specifications at Geneart (Regensburg, Germany).

DNA Sequence Determination

DNA sequences were determined by double strand sequencing performed atMediGenomix GmbH (Martinsried, Germany) or Sequiserve GmbH(Vaterstetten, Germany).

DNA and Protein Sequence Analysis and Sequence Data Management

The GCG's (Genetics Computer Group, Madison, Wis.) software packageversion 10.2 and Infomax's Vector NT1 Advance suite version 8.0 was usedfor sequence creation, mapping, analysis, annotation and illustration.Expression vectors

For the expression of the described antibodies variants of expressionplasmids for transient expression (e.g. in HEK293 EBNA or HEK293-F)cells based either on a cDNA organization with a CMV-Intron A promoteror on a genomic organization with a CMV promoter were applied.

Beside the antibody expression cassette the vectors contained:

an origin of replication which allows replication of this plasmid in E.coli, and

a β-lactamase gene which confers ampicillin resistance in E. coli.

The transcription unit of the antibody gene is composed of the followingelements:

unique restriction site(s) at the 5′ end

the immediate early enhancer and promoter from the humancytomegalovirus,

followed by the Intron A sequence in the case of the cDNA organization,

a 5′-untranslated region of a human antibody gene,

a immunoglobulin heavy chain signal sequence,

the human antibody chain (wildtype or with domain exchange) either ascDNA or as genomic organization with an the immunoglobulin exon-intronorganization

a 3′ untranslated region with a polyadenylation signal sequence, and

unique restriction site(s) at the 3′ end.

The fusion genes comprising the described antibody chains as describedbelow were generated by PCR and/or gene synthesis and assembled withknown recombinant methods and techniques by connection of the accordingnucleic acid segments e.g. using unique restriction sites in therespective vectors. The subcloned nucleic acid sequences were verifiedby DNA sequencing. For transient transfections larger quantities of theplasmids were prepared by plasmid preparation from transformed E. colicultures (Nucleobond AX, Macherey-Nagel).

Cell Culture Techniques

Standard cell culture techniques were used as described in CurrentProtocols in Cell Biology (2000), Bonifacino, J. S., Dasso, M., Harford,J. B., Lippincott-Schwartz, J. and Yamada, K. M. (eds.), John Wiley &Sons, Inc.

Bispecific antibodies were expressed by transient co-transfection of therespective expression plasmids in adherently growing HEK293-EBNA or inHEK29-F cells growing in suspension as described below.

Transient Transfections in HEK293-EBNA System

Bispecific antibodies were expressed by transient co-transfection of therespective expression plasmids (e.g. encoding the heavy and modifiedheavy chain, as well as the corresponding light and modified lightchain) in adherently growing HEK293-EBNA cells (human embryonic kidneycell line 293 expressing Epstein-Barr-Virus nuclear antigen; Americantype culture collection deposit number ATCC #CRL-10852, Lot. 959 218)cultivated in DMEM (Dulbecco's modified Eagle's medium, Gibco)supplemented with 10% Ultra Low IgG FCS (fetal calf serum, Gibco), 2 mML-Glutamine (Gibco), and 250 μg/ml Geneticin (Gibco). For transfectionFuGENE™ 6 Transfection Reagent (Roche Molecular Biochemicals) was usedin a ratio of FuGENE™ reagent (μl) to DNA (μg) of 4:1 (ranging from 3:1to 6:1). Proteins were expressed from the respective plasmids using amolar ratio of (modified and wildtype) light chain and heavy chainencoding plasmids of 1:1 (equimolar) ranging from 1:2 to 2:1,respectively. Cells were feeded at day 3 with L-Glutamine ad 4 mM,Glucose [Sigma] and NAA [Gibco]. Bispecific antibody containing cellculture supernatants were harvested from day 5 to 11 after transfectionby centrifugation and stored at −20° C. General information regardingthe recombinant expression of human immunoglobulins in e.g. HEK293 cellsis given in: Meissner, P. et al., Biotechnol. Bioeng. 75 (2001) 197-203.

Transient Transfections in HEK293-F System

Bispecific antibodies were generated by transient transfection of therespective plasmids (e.g. encoding the heavy and modified heavy chain,as well as the corresponding light and modified light chain) using theHEK293-F system (Invitrogen) according to the manufacturer'sinstruction. Briefly, HEK293-F cells (Invitrogen) growing in suspensioneither in a shake flask or in a stirred fermenter in serumfree FreeStyle293 expression medium (Invitrogen) were transfected with a mix of thefour expression plasmids and 293fectin or fectin (Invitrogen). For 2 Lshake flask (Corning) HEK293-F cells were seeded at a density of 1.0E*6cells/mL in 600 mL and incubated at 120 rpm, 8% CO₂. The day after thecells were transfected at a cell density of ca. 1.5E*6 cells/mL with ca.42 mL mix of A) 20 mL Opti-MEM (Invitrogen) with 600 μg total plasmidDNA (1 μg/mL) encoding the heavy or modified heavy chain, respectivelyand the corresponding light chain in an equimolar ratio and B) 20 mlOpti-MEM+1.2 mL 293 fectin or fectin (2 μl/mL). According to the glucoseconsumption glucose solution was added during the course of thefermentation. The supernatant containing the secreted antibody washarvested after 5-10 days and antibodies were either directly purifiedfrom the supernatant or the supernatant was frozen and stored.

Protein Determination

The protein concentration of purified antibodies and derivatives wasdetermined by determining the optical density (OD) at 280 nm, using themolar extinction coefficient calculated on the basis of the amino acidsequence according to Pace, C. N., et. al., Protein Science, 1995, 4,2411-1423.

Antibody Concentration Determination in Supernatants

The concentration of antibodies and derivatives in cell culturesupernatants was estimated by immunoprecipitation with Protein AAgarose-beads (Roche). 60 μL Protein A Agarose beads are washed threetimes in TBS-NP40 (50 mM Tris, pH 7.5, 150 mM NaCl, 1% Nonidet-P40).Subsequently, 1-15 mL cell culture supernatant were applied to theProtein A Agarose beads pre-equilibrated in TBS-NP40. After incubationfor at 1 h at room temperature the beads were washed on anUltrafree-MC-filter column (Amicon] once with 0.5 mL TBS-NP40, twicewith 0.5 mL 2× phosphate buffered saline (2×PBS, Roche) and briefly fourtimes with 0.5 mL 100 mM Na-citrate pH 5.0. Bound antibody was eluted byaddition of 35 μl NuPAGE® LDS Sample Buffer (Invitrogen). Half of thesample was combined with NuPAGE® Sample Reducing Agent or leftunreduced, respectively, and heated for 10 min at 70° C. Consequently,5-30 μl were applied to an 4-12% NuPAGE® Bis-Tris SDS-PAGE (Invitrogen)(with MOPS buffer for non-reduced SDS-PAGE and MES buffer with NuPAGE®Antioxidant running buffer additive (Invitrogen) for reduced SDS-PAGE)and stained with Coomassie Blue.

The concentration of antibodies and derivatives in cell culturesupernatants was quantitatively measured by affinity HPLCchromatography. Briefly, cell culture supernatants containing antibodiesand derivatives that bind to Protein A were applied to an AppliedBiosystems Poros A/20 column in 200 mM KH2PO4, 100 mM sodium citrate, pH7.4 and eluted from the matrix with 200 mM NaCl, 100 mM citric acid, pH2.5 on an Agilent HPLC 1100 system. The eluted protein was quantified byUV absorbance and integration of peak areas. A purified standard IgG1antibody served as a standard.

Alternatively, the concentration of antibodies and derivatives in cellculture supernatants was measured by Sandwich-IgG-ELISA. Briefly,StreptaWell High Bind Strepatavidin A-96 well microtiter plates (Roche)were coated with 100 μL/well biotinylated anti-human IgG capturemolecule F(ab′)2<h-Fcγ> BI (Dianova) at 0.1 μg/mL for 1 h at roomtemperature or alternatively over night at 4° C. and subsequently washedthree times with 200 μL/well PBS, 0.05% Tween (PBST, Sigma). 100 μL/wellof a dilution series in PBS (Sigma) of the respective antibodycontaining cell culture supernatants was added to the wells andincubated for 1-2 h on a microtiterplate shaker at room temperature. Thewells were washed three times with 200 μL/well PBST and bound antibodywas detected with 100 μl F(ab′)2<hFcγ>POD (Dianova) at 0.1 μg/mL asdetection antibody for 1-2 h on a microtiterplate shaker at roomtemperature. Unbound detection antibody was washed away three times with200 μL/well PBST and the bound detection antibody was detected byaddition of 100 μL ABTS/well. Determination of absorbance was performedon a Tecan Fluor Spectrometer at a measurement wavelength of 405 nm(reference wavelength 492 nm).

Protein Purification

Proteins were purified from filtered cell culture supernatants referringto standard protocols. In brief, antibodies were applied to a Protein ASepharose column (GE healthcare) and washed with PBS. Elution ofantibodies was achieved at pH 2.8 followed by immediate neutralizationof the sample. Aggregated protein was separated from monomericantibodies by size exclusion chromatography (Superdex 200, GEHealthcare) in PBS or in 20 mM Histidine, 150 mM NaCl pH 6.0. Monomericantibody fractions were pooled, concentrated if required using e.g. aMILLIPORE Amicon Ultra (30 MWCO) centrifugal concentrator, frozen andstored at −20° C. or −80° C. Part of the samples were provided forsubsequent protein analytics and analytical characterization e.g. bySDS-PAGE, size exclusion chromatography or mass spectrometry.

SDS-PAGE

The NuPAGE® Pre-Cast gel system (Invitrogen) was used according to themanufacturer's instruction. In particular, 10% or 4-12% NuPAGE® Novex®Bis-TRIS Pre-Cast gels (pH 6.4) and a NuPAGE® MES (reduced gels, withNuPAGE® Antioxidant running buffer additive) or MOPS (non-reduced gels)running buffer was used.

Analytical Size Exclusion Chromatography

Size exclusion chromatography for the determination of the aggregationand oligomeric state of antibodies was performed by HPLC chromatography.Briefly, Protein A purified antibodies were applied to a Tosoh TSKgelG3000SW column in 300 mM NaCl, 50 mM KH2PO4/K2HPO4, pH 7.5 on an AgilentHPLC 1100 system or to a Superdex 200 column (GE Healthcare) in 2×PBS ona Dionex HPLC-System. The eluted protein was quantified by UV absorbanceand integration of peak areas. BioRad Gel Filtration Standard 151-1901served as a standard.

Mass Spectrometry

The total deglycosylated mass of crossover antibodies was determined andconfirmed via electrospray ionization mass spectrometry (ESI-MS).Briefly, 100 μg purified antibodies were deglycosylated with 50 mUN-Glycosidase F (PNGaseF, ProZyme) in 100 mM KH2PO4/K2HPO4, pH 7 at 37°C. for 12-24 h at a protein concentration of up to 2 mg/ml andsubsequently desalted via HPLC on a Sephadex G25 column (GE Healthcare).The mass of the respective heavy and light chains was determined byESI-MS after deglycosylation and reduction. In brief, 50 μg antibody in115 μl were incubated with 60 μl 1M TCEP and 50 μl 8 MGuanidine-hydrochloride subsequently desalted. The total mass and themass of the reduced heavy and light chains was determined via ESI-MS ona Q-Star Elite MS system equipped with a NanoMate source.

IGF-1R ECD Binding ELISA

The binding properties of the generated antibodies were evaluated in anELISA assay with the IGF-1R extracellular domain (ECD). For this sakethe extracellular domain of IGF-1R (residues 1-462) comprising thenatural leader sequence and the LI-cysteine rich-12 domains of the humanIGF-1R ectodomain of the alpha chain (according to the McKern et al.,1997; Ward et al., 2001) fused to an N-terminal His-Streptavidin bindingpeptide-tag (His-SBP) was cloned into a pcDNA3 vector derivative andtransiently expressed in HEK293F cells. The protein sequence of theIGF-1R-His-SBP ECD is given in SEQ ID NO: 5. StreptaWell High BindStrepatavidin A-96 well microtiter plates (Roche) were coated with 100μL/well cell culture supernatant containing soluble IGF-1R-ECD-SBPfusion protein over night at 4° C. and washed three times with 200μL/well PBS, 0.05% Tween (PBST, Sigma). Subsequently, 100 μL/well of adilution series of the respective antibody and as a reference wildtype<IGF-1R> antibody in PBS (Sigma) including 1% BSA (fraction V, Roche)was added to the wells and incubated for 1-2 h on a microtiterplateshaker at room temperature. For the dilution series either the sameamount of purified antibody as a reference or supernatants fromtransient transfection in HEK293E (HEK293F) normalized bySandwich-IgG-ELISA for the same antibody concentration were applied tothe wells. The wells were washed three times with 200 μL/well PBST andbound antibody was detected with 100 μL/well F(ab′)2<hFcγ>POD (Dianova)at 0.1 μg/mL as detection antibody for 1-2 h on a microtiterplate shakerat room temperature. Unbound detection antibody was washed away threetimes with 200 μL/well PBST and the bound detection antibody wasdetected by addition of 100 μL ABTS/well. Determination of absorbancewas performed on a Tecan Fluor Spectrometer at a measurement wavelengthof 405 nm (reference wavelength 492 nm).

IGF-1R ECD Biacore

Binding of the generated antibodies to human IGF-1R ECD was alsoinvestigated by surface plasmon resonance using a BIACORE T100instrument (GE Healthcare Biosciences AB, Uppsala, Sweden). Briefly, foraffinity measurements Goat-Anti-Human IgG, JIR 109-005-098 antibodieswere immobilized on a CM5 chip via amine coupling for presentation ofthe antibodies against human IGF-1R ECD-Fc tagged. Binding was measuredin HBS buffer (HBS-P (10 mM HEPES, 150 mM NaCl, 0.005% Tween 20, ph7.4), 25° C. IGF-1R ECD (R&D Systems or in house purified) was added invarious concentrations in solution. Association was measured by anIGF-1R ECD injection of 80 seconds to 3 minutes; dissociation wasmeasured by washing the chip surface with HBS buffer for 3-10 minutesand a KD value was estimated using a 1:1 Langmuir binding model. Due tolow loading density and capturing level of <IGF-1R> antibodiesmonovalent IGF-1R ECD binding was obtained. Negative control data (e.g.buffer curves) were subtracted from sample curves for correction ofsystem intrinsic baseline drift and for noise signal reduction. BiacoreT100 Evaluation Software version 1.1.1 was used for analysis ofsensorgrams and for calculation of affinity data (FIG. 18).

ANGPT2 Binding ELISA

The binding properties of the generated antibodies were evaluated in anELISA assay with full-length ANGPT2-His protein (R&D Systems). For thissake Falcon polystyrene clear enhanced microtiter plates were coatedwith 100 μl 1 μg/mL recombinant human ANGPT2 (R&D Systems, carrier-free)in PBS for 2 h at room temperature or over night at 4° C. The wells werewashed three times with 300% PBST (0.2% Tween 20) and blocked with 200μl 2% BSA 0.1% Tween 20 for 30 min at room temperature and subsequentlywashed three times with 300% PBST. 100 μL/well of a dilution series ofpurified <ANGPT2> VL-VH/CL-CH1 exchange antibody and as a referencewildtype <ANGPT2> antibody in PBS (Sigma) were added to the wells andincubated for 1 h on a microtiterplate shaker at room temperature. Thewells were washed three times with 300 μl PBST (0.2% Tween 20) and boundantibody was detected with 100 μL/well 0.1 μg/ml F(ab′) <hk>POD (BiozolCat. No. 206005) or 100 μL/well 0.1 μg/ml F(ab′) <hFcγ>POD (Immunoresearch) in 2% BSA 0.1% Tween 20 in 2% BSA 0.1% Tween 20 as detectionantibody for 1 h on a microtiterplate shaker at room temperature.Unbound detection antibody was washed away three times with 300 μL/wellPBST and the bound detection antibody was detected by addition of 100 μLABTS/well. Determination of absorbance was performed on a Tecan FluorSpectrometer at a measurement wavelength of 405 nm (reference wavelength492 nm).

ANGPT2 Binding BIACORE

Binding of the generated antibodies to human ANGPT2 was alsoinvestigated by surface plasmon resonance using a BIACORE T100instrument (GE Healthcare Biosciences AB, Uppsala, Sweden). Briefly, foraffinity measurements goat<hIgG-Fcg> polyclonal antibodies wereimmobilized on a CM5 or CM4 chip via amine coupling for presentation ofthe antibodies against human ANGPT2. Binding was measured in HBS buffer(HBS-P (10 mM HEPES, 150 mM NaCl, 0.005% Tween 20, ph 7.4), with orwithout 5 mM Ca2+, 25° C. Purified ANGPT2-His (R&D Systems or in housepurified) was added in various concentrations in solution. Associationwas measured by an ANGPT2-injection of 3 minutes; dissociation wasmeasured by washing the chip surface with HBS buffer for 3 to 5 minutesand a KD value was estimated using a 1:1 Langmuir binding model. Due toheterogenity of the ANGPT2 preparation no 1:1 binding could be observed;KD values are thus only relative estimations. Negative control data(e.g. buffer curves) were subtracted from sample curves for correctionof system intrinsic baseline drift and for noise signal reduction.Biacore T100 Evaluation Software version 1.1.1 was used for analysis ofsensorgrams and for calculation of affinity data (FIG. 19)

Inhibition of hANGPT2 Binding to Tie-2-ECD (ELISA)

To test the ability of ANGPT2 antibodies to interfere with Tie2 bindingthe following ELISA was set up. The test was performed on 384 wellmicrotiter plates (MicroCoat, DE, Cat. No. 464718) at RT. After eachincubation step plates were washed 3 times with PBST. At the beginning,plates were coated with 0.5 μg/ml Tie-2 protein (R&D Systems, UK, Cat.No. 313-TI) for at least 2 hours (h). Thereafter the wells were blockedwith PBS supplemented with 0.2% Tween-20 and 2% BSA (Roche DiagnosticsGmbH, DE) for 1 h. Dilutions of purified antibodies in PBS wereincubated together with 0.2 μg/ml huANGPT2 (R&D Systems, UK, Cat. No.623-AN) for 1 h at RT. After washing a mixture of 0.5 μg/ml biotinylatedanti-ANGPT2 clone BAM0981 (R&D Systems, UK) and 1:3000 dilutedstreptavidin HRP (Roche Diagnostics GmbH, DE, Cat. No. 11089153001) wasadded for 1 h. Thereafter the plates were washed 6 times with PBST.Plates were developed with freshly prepared ABTS reagent (RocheDiagnostics GmbH, DE, buffer #204 530 001, tablets #11 112 422 001) for30 minutes at RT. Absorbance was measured at 405 nm.

ANGPT2-VEGF Bridging ELISA

The binding properties of the generated bispecific antibodies wasevaluated in an ELISA assay with immobilized full-length VEGF165-Hisprotein (R&D Systems) and human ANGPT2-His protein (R&D Systems) fordetection of simultaneously bound bispecific antibody. Only a bispecificantibody is able to bind simultaneously to VEGF and ANGPT2 and thusbridge the two antigens whereas monospecific “standard” IgG1 antibodiesshould not be capable of simultaneously binding to VEGF and ANGPT2. Forthis sake Falcon polystyrene clear enhanced microtiter plates werecoated with 100 μl 2 μg/mL recombinant human VEGF165 (R&D Systems) inPBS for 2 h at room temperature or over night at 4° C. The wells werewashed three times with 300 μl PBST (0.2% Tween 20) and blocked with 200μl 2% BSA 0.1% Tween 20 for 30 min at room temperature and subsequentlywashed three times with 300 μl PBST. 100 μL/well of a dilution series ofpurified bispecific antibody and control antibodies in PBS (Sigma) wasadded to the wells and incubated for 1 h on a microtiterplate shaker atroom temperature. The wells were washed three times with 300%1 PBST(0.2% Tween 20) and bound antibody was detected by addition of 100 μl0.5 μg/ml human ANGPT2-His (R&D Systems) in PBS. The wells were washedthree times with 300%1 PBST (0.2% Tween 20) and bound ANGPT2 wasdetected with 100 μl 0.5 μg/mL <ANGPT2> mIgG1-Biotin antibody (BAM0981,R&D Systems) for 1 h at room temperature. Unbound detection antibody waswashed away with three times 300 μl PBST (0.2% Tween 20) and boundantibody was detected by addition of 100 μl 1:2000 Streptavidin-PODconjugate (Roche Diagnostics GmbH, Cat. No. 11089153) 1:4 diluted inblocking buffer for 1 h at room temperature. Unbound Streptavidin-PODconjugate was washed away with three-six times 300 μl PBST (0.2% Tween20) and bound Strepatavidin-POD conjugate was detected by addition of100 μL ABTS/well. Determination of absorbance was performed on a TecanFluor Spectrometer at a measurement wavelength of 405 nm (referencewavelength 492 nm) (FIG. 20).

Biacore Assay to Detect Simultaneous Binding of Bispecific Antibodies toVEGF and ANGPT2

In order to further corroborate the data from the bridging ELISA anadditional assay was established using surface plasmon resonancetechnology on a Biacore T100 instrument to confirm simultaneous bindingto VEGF and ANGPT2 according to the following protocol. Data wereanalyzed using the T100 software package: ANGPT2 was captured with acapture level of 2000-1700 RU via a PentaHisAntibody (SEQ ID NO: 19)(PentaHis-Ab (SEQ ID NO: 19) BSA-free, Qiagen No. 34660) that wasimmobilized on a CM5 chip via amincoupling (BSA-free). HBS-N bufferserved as running buffer, activation was done by mixture of EDC/NHS. ThePentaHis-Ab (SEQ ID NO: 19) BSA-free Capture-Antibody was diluted incoupling buffer NaAc, pH 4.5, c=30 μg/mL, finally still activatedcarboxyl groups were blocked by injection of 1 M Ethanolamin, liganddensities of 5000 and 17000 RU were tested. ANGPT2 with a concentrationof 500 nM was captured by the PentaHis-Ab (SEQ ID NO: 19) at a flow of 5μL/min diluted with running buffer+1 mg/mL BSA. Subsequently, bispecificantibody binding to ANGPT2 and to VEGF was demonstrated by incubationwith rhVEGF. For this sake, bispecific antibody was bound to ANGPT2 at aflow of 50 μL/min and a concentration of 100-500 nM, diluted withrunning buffer+1 mg/mL BSA and simultaneous binding was detected byincubation with VEGF (rhVEGF, R&D-Systems Cat.-No. 293-VE) in PBS+0.005%(v/v) Tween20 running buffer+1 mg/ml BSA at a flow of 50 μL/min and aVEGF concentration of 100-150 nM. Association time 120 sec, dissociationtime 1200 sec. Regeneration was done at a flow of 50 μL/min with 2×10 mMGlycin pH 2.0 and a contact time of 60 sec. Sensorgrams were correctedusing the usual double referencing (control reference: binding ofbispecific antibody and rhVEGF to capture molecule PentaHisAb (SEQ IDNO: 19)). Blanks for each Ab were measured with rhVEGF concentration“0”. A scheme of the Biacore assay is shown in FIG. 21. Sensorgrams werecorrected using the usual double referencing (control reference: bindingof bispecific Ab and rhVEGF to capture molecule PentaHisAb (SEQ ID NO:19). Blanks for each Ab were measured with rhVEGF concentration “0”.

Examples 1

Production, expression, purification and characterization ofmonospecific, bivalent <IGF-1R> antibody, wherein the variable domainsVL and VH are replaced by each other, and wherein the constant domainsCL and CH1 are replaced by each other (abbreviated herein as <IGF-1R>VL-VH/CL-CH1 exchange antibody).

Example 1A

Making of the expression plasmids for the monospecific, bivalent<IGF-1R> VL-VH/CL-CH1 exchange antibody.

The sequences for the heavy and light chain variable domains of themonospecific, bivalent Insulin-like Growth Factor 1 Receptor <IGF-1R>VL-VH/CL-CH1 exchange antibody including the respective leader sequencesdescribed in this example are derived from wild type <IGF-1R> antibodyheavy chain (SEQ ID NO: 1, plasmid 4843-pUC-HC-IGF-1R) and a light chain(SEQ ID NO: 2, plasmid 4842-pUC-LC-IGF-1R) described in WO 2005/005635,and the heavy and light chain constant domains are derived from a humanantibody (C-kappa and IgG1).

The gene segments encoding the <IGF-1R> antibody leader sequence, lightchain variable domain (VL) and the human kappa-light chain constantdomain (CL) were joined and fused to the 5′-end of the Fc domains of thehuman γ1-heavy chain constant domains (Hinge-CH2-CH3). The DNA codingfor the respective fusion protein resulting from the exchange of VH andCH1 domains by VL and CL domains was generated by gene synthesis and isdenoted <IGF-1R> HC* (Heavy Chain*) (SEQ ID NO: 3) in the following.

The gene segments for the <IGF-1R> antibody leader sequence, heavy chainvariable domain (VH) and the human γ1-heavy chain constant domains (CH1)were joined as independent chain. The DNA coding for the respectivefusion protein resulting from the exchange of VL and CL domains by VHand CH1 domains was generated by gene synthesis and is denoted <IGF-1R>LC* (Light Chain)* (SEQ ID NO: 4) in the following.

FIG. 5 and FIG. 6 show a schematic view of the protein sequence of themodified <IGF-1R> HC* heavy chain* and the modified <IGF-1R> LC* lightchain*.

In the following the respective expression vectors are brieflydescribed:

Vector DW047-pUC-HC*-IGF-1R

Vector DW047-pUC-HC*-IGF-1R is an expression plasmid e.g. for transientexpression of a <IGF-1R> heavy chain* HC* (cDNA organized expressioncassette; with CMV-Intron A in HEK293 (EBNA) cells or for stableexpression in CHO cells.

Beside the <IGF-1R> HC* expression cassette this vector contains:

an origin of replication from the vector pUC18 which allows replicationof this plasmid in E. coli, and

a β-lactamase gene which confers ampicillin resistance in E. coli.

The transcription unit of the <IGF-1R> HC* gene is composed of thefollowing elements:

the unique AscI restriction site at the 5′-end

the immediate early enhancer and promoter from the humancytomegalovirus,

followed by the Intron A sequence,

a 5′-untranslated region of a human antibody gene,

a immunoglobulin light chain signal sequence,

the human <IGF-1R> mature HC* chain encoding a fusion of the human lightchain variable domain (VL) and the human kappa-light chain constantdomain (CL) fused to the 5′-end of the Fc domains of the human γ1-heavychain constant domains (Hinge-CH2-CH3).

a 3′ untranslated region with a polyadenylation signal sequence, and

the unique restriction site SgrAI at the 3′-end.

The plasmid map of the <IGF-1R> HC* expression vectorDW047-pUC-HC*-IGF-1R is shown in FIG. 7. The amino acid sequence of the<IGF-1R> HC* (including signal sequence) is given in SEQ ID NO: 3.

Vector DW048-pUC-LC*-IGF-1R

Vector DW048-pUC-LC*-IGF-1R is an expression plasmid e.g. for transientexpression of a VL-VH/CL-CH1 exchange <IGF-1R> light chain* LC* (cDNAorganized expression cassette; with CMV-Intron A in HEK293 (EBNA) cellsor for stable expression in CHO cells.

Beside the <IGF-1R> LC* expression cassette this vector contains:

an origin of replication from the vector pUC18 which allows replicationof this plasmid in E. coli, and

a β-lactamase gene which confers ampicillin resistance in E. coli.

The transcription unit of the <IGF-1R> LC* gene is composed of thefollowing elements:

the unique restriction site Sse8387I at the 5′ end

the immediate early enhancer and promoter from the humancytomegalovirus,

followed by the Intron A sequence,

a 5′-untranslated region of a human antibody gene,

a immunoglobulin heavy chain signal sequence,

the human <IGF-1R> antibody mature LC* chain encoding a fusion of thehuman heavy chain variable domain (VH) and the human γ1-heavy chainconstant domains (CH1).

a 3′ untranslated region with a polyadenylation signal sequence, and

the unique restriction sites SalI and FseI at the 3′-end.

The plasmid map of the light chain* <IGF-1R> LC* expression vectorDW048-pUC-LC*-IGF-1R is shown in FIG. 8. The amino acid sequence of the<IGF-1R> LC* (including signal sequence) is given in SEQ ID NO: 4.

Plasmids DW047-pUC-HC*-IGF-1R and DW048-pUC-LC*-IGF-1R can be used fortransient or stable co-transfections e.g. into HEK293, HEK293 EBNA orCHO cells (2-vector system). For comparative reasons the wildtype<IGF-1R> antibody was transiently expressed from plasmids4842-pUC-LC-IGF-1R (SEQ ID NO: 2) and 4843-pUC-HC-IGF-1R (SEQ ID NO: 1)analogous to the ones described in this example.

In order to achieve higher expression levels in transient expressions inHEK293 EBNA cells the <IGF-1R> HC* expression cassette can be sub-clonedvia AscI, SgrAI sites and the <IGF-1R> LC* expression cassette can besub-cloned via Sse8387I and FseI sites into the 4700 pUC-Hyg_OriPexpression vector containing

an OriP element, and

a hygromycine resistance gene as a selectable marker.

Heavy and light chain transcription units can either be sub-cloned intotwo independent 4700-pUC-Hyg-OriP vectors for co-transfection (2-vectorsystem) or they can be cloned into one common 4700-pUC-Hyg-OriP vector(1-vector system) for subsequent transient or stable transfections withthe resulting vectors. FIG. 9 shows a plasmid map of the basic vector4700-pUC-OriP.

Example 1B Making of the Monospecific, Bivalent <IGF-1R> VL-VH/CL-CH1Exchange Antibody Expression Plasmids

The <IGF-1R> fusion genes (HC* and LC* fusion genes) comprising theexchanged Fab sequences of the wildtype <IGF-1R> antibody were assembledwith known recombinant methods and techniques by connection of theaccording nucleic acid segments.

The nucleic acid sequences encoding the IGF-1R HC* and LC* were eachsynthesized by chemical synthesis and subsequently cloned into apPCRScript (Stratagene) based pGA4 cloning vector at Geneart(Regensburg, Germany). The expression cassette encoding the IGF-1R HC*was ligated into the respective E. coli plasmid via PvuII and BmgBIrestriction sites resulting in the final vector DW047-pUC-HC*-IGF-1R;the expression cassette encoding the respective IGF-1R LC* was ligatedinto the respective E. coli plasmid via PvuII and SalI restriction sitesresulting in the final vector DW048-pUC-LC*-IGF-1R. The subclonednucleic acid sequences were verified by DNA sequencing. For transientand stable transfections larger quantities of the plasmids were preparedby plasmid preparation from transformed E. coli cultures (Nucleobond AX,Macherey-Nagel).

Example 1C Transient Expression of Monospecific, Bivalent <IGF-1R>VL-VH/CL-CH1 Exchange Antibody in HEK293 EBNA Cells

Recombinant <IGF-1R> VL-VH/CL-CH1 exchange antibody was expressed bytransient co-transfection of plasmids DW047-pUC-HC*-IGF-1R andDW048-pUC-LC*-IGF-1R in adherently growing HEK293-EBNA cells (humanembryonic kidney cell line 293 expressing Epstein-Barr-Virus nuclearantigen; American type culture collection deposit number ATCC #CRL-10852, Lot. 959 218) cultivated in DMEM (Dulbecco's modified Eagle'smedium, Gibco) supplemented with 10% Ultra Low IgG FCS (fetal calfserum, Gibco), 2 mM L-Glutamine (Gibco), and 250 μg/ml Geneticin(Gibco). For transfection FuGENE™ 6 Transfection Reagent (RocheMolecular Biochemicals) was used in a ratio of FuGENE™ reagent (μl) toDNA (μg) of 4:1 (ranging from 3:1 to 6:1). Light and heavy chainplasmids encoding <IGF-1R> HC* and LC* (plasmids DW047-pUC-HC*-IGF-1Rand DW048-pUC-LC*-IGF-1R) were expressed from two different plasmidsusing a molar ratio of light chain to heavy chain encoding plasmids of1:1 (equimolar) ranging from 1:2 to 2:1, respectively. Cells were feededat day 3 with L-Glutamine ad 4 mM, Glucose [Sigma] and NAA [Gibco].<IGF-1R> VL-VH/CL-CH1 exchange antibody containing cell culturesupernatants were harvested from day 5 to 11 after transfection bycentrifugation and stored at −20° C. General information regarding therecombinant expression of human immunoglobulins in e.g. HEK293 cells isgiven in: Meissner, P. et al., Biotechnol. Bioeng. 75 (2001) 197-203.

Example 1D Immunoprecipitation of Monospecific, Bivalent <IGF-1R>VL-VH/CL-CH1 Exchange Antibody

Monospecific, bivalent <IGF-1R> VL-VH/CL-CH1 exchange antibody wasisolated from cell culture supernatants (example 1C) byimmunoprecipitation with Protein A Agarose-beads (Roche). 60 μL ProteinA Agarose beads were washed three times in TBS-NP40 (50 mM Tris, pH 7.5,150 mM NaCl, 1% Nonidet-P40). Subsequently, 1-15 mL cell culturesupernatant were applied to the Protein A Agarose beads pre-equilibratedin TBS-NP40. After incubation for at 1 h at room temperature the beadswere washed on an Ultrafree-MC-filter column (Amicon] once with 0.5 mLTBS-NP40, twice with 0.5 mL 2× phosphate buffered saline (2×PBS, Roche)and briefly four times with 0.5 mL 100 mM Na-citrate pH 5.0. Boundantibody was eluted by addition of 35 μl NuPAGE® LDS Sample Buffer(Invitrogen). Half of the sample was combined with NuPAGE® SampleReducing Agent or left unreduced, respectively, and heated for 10 min at70° C. Consequently, 20 μl were applied to an 4-12% NuPAGE® Bis-TrisSDS-PAGE (Invitrogen) (with MOPS buffer for non-reduced SDS-PAGE and MESbuffer with NuPAGE® Antioxidant running buffer additive (Invitrogen) forreduced SDS-PAGE) and stained with Coomassie Blue. FIG. 10 shows theSDS-PAGE from a typical immunoprecipitation experiment. Themonospecific, bivalent <IGF-1R> VL-VH/CL-CH1 exchange antibody behaveslike a typical IgG1 antibody with a ca. 25 kDa band corresponding to theLC* light chain and a 50 kDa band corresponding to the respective HC*heavy chain. In the unreduced state, a band at ca. 150 kDa can beobserved for the complete antibody.

Example 1E Purification of Monospecific, Bivalent <IGF-1R> VL-VH/CL-CH1Exchange Antibody and Confirmation of Identity by Mass Spectrometry

The expressed and secreted monospecific, bivalent <IGF-1R> VL-VH/CL-CH1exchange antibody was purified from filtered cell culture supernatantsby Protein A affinity chromatography according to known standardmethods. In brief, the <IGF-1R> VL-VH/CL-CH1 exchange antibodycontaining cell culture supernatants from transient transfections wereclarified by centrifugation (10,000 g for 10 minutes) and filtrationthrough a 0.45 μm filter and applied to a Protein A HiTrap MabSelectXtra column (GE Healthcare) equilibrated with PBS buffer (10 mM Na2HPO4,1 mM KH2PO4, 137 mM NaCl and 2.7 mM KCl, pH 7.4). Unbound proteins werewashed out with PBS equilibration buffer followed by 0.1 M sodiumcitrate buffer, pH 5.5 and washed with PBS. Elution of <IGF-1R>VL-VH/CL-CH1 exchange antibody was achieved with 100 mM sodium citrate,pH 2.8 followed by immediate neutralization of the sample with 300 μl 2M Tris pH 9.0 per 2 ml fraction. Aggregated protein was separated frommonomeric antibodies by size exclusion chromatography on a HiLoad 26/60Superdex 200 prep grade column (GE Healthcare) in 20 mM Histidine, 150mM NaCl pH 6.0 and monomeric antibody fractions were subsequentlyconcentrated using a MILLIPORE Amicon Ultra-15 centrifugal concentrator.<IGF-1R> VL-VH/CL-CH1 exchange antibody was frozen and stored at −20° C.or −80° C. The integrity of the <IGF-1R> VL-VH/CL-CH1 exchange wasanalyzed by SDS-PAGE in the presence and absence of a reducing agent andsubsequent staining with Coomassie brilliant blue as described inExample 1D. The purified <IGF-1R> VL-VH/CL-CH1 exchange antibody behaveslike a typical IgG1 antibody with a ca. 25 kDa band corresponding to theLC* light chain and a 50 kDa band corresponding to the respective HC*heavy chain. In the unreduced state a band at ca. 150 kDa can beobserved for the complete antibody. Aggregation and oligomeric state ofthe <IGF-1R>

VL-VH/CL-CH1 exchange antibody was analyzed by analytical size exclusionchromatography and showed that the purified Fab crossover antibody wasin a monomeric state. Characterized samples were provided for subsequentprotein analytics and functional characterization. ESI mass spectrometryconfirmed the theoretical molecular mass (calculated without C-terminallysine residue of the heavy chain and with a C-terminal pyroglutamateresidue of the light chain) of the completely deglycosylated <IGF-1R>VL-VH/CL-CH1 exchange antibody as main species. (FIG. 22)

Example 1F Analysis of the IGF-1R Binding Properties of Monospecific,Bivalent <IGF-1R> VL-VH/CL-CH1 Exchange Antibody in an IGF-1R ECDBinding ELISA and by Biacore

The binding properties of monospecific, bivalent <IGF-1R> VL-VH/CL-CH1exchange antibody were evaluated in an ELISA assay with the IGF-1Rextracellular domain (ECD) as described above. FIG. 11 shows thenormalized results from a titration of <IGF-1R> VL-VH/CL-CH1 exchangeantibody and wildtype <IGF-1R> antibody from transient transfectionsupernatants in HEK293E by Sandwich-IgG-ELISA. It can be clearly seenthat <IGF-1R> VL-VH/CL-CH1 exchange antibody is functional and showscomparable binding characteristics as the wildtype <IGF-1R> antibody andthus appears fully functional. The minor observed differences lie withinthe error of the method and might e.g. result from minor variances inprotein concentration.

These findings were corroborated by Biacore data with the respectivepurified antibodies that showed that monospecific, bivalent <IGF-1R>VL-VH/CL-CH1 exchange antibody had a comparable affinity and bindingkinetics for IGF-1R ECD as the original wildtype antibody within theerror of the method. The kinetic data are given in the following table:

ka [1/Ms] kd [1/s] K(D) [M] wildtype <IGF-1R> antibody 3.18E+06 5.521E−31.74E−09 <IGF-1R> VL-VH/CL-CH1 2.65E+06 6.258E−3 2.36E−09 exchangeantibody

Example 1G Analysis of the IGF-1R Binding Properties of Monospecific,Bivalent <IGF-1R> VL-VH/CL-CH1 Exchange Antibody by FACS with IGF-1ROver-Expressing 124 Cells

In order to confirm the binding activity of <IGF-1R> VL-VH/CL-CH1exchange antibody binding to the IGF-1R over-expressed on the surface of124 cells (NIH3T3 cells expressing recombinant human IGF-1R, Roche) isstudied by FACS. Briefly, 5×10E5 I24 cells per FACS tube are incubatedwith a dilution of purified <IGF-1R> VL-VH/CL-CH1 exchange antibody andwildtype <IGF-1R> antibody as a reference and incubated on ice for 1 h.Unbound antibody is washed away with 4 ml ice cold PBS (Gibco)+2% FCS(Gibco). Subsequently, cells are centrifuged (5 min at 400 g) and boundantibody is detected with F(ab′)2 <hFcγ>PE conjugate (Dianova) on icefor 1 h protected from light. Unbound detection antibody is washed awaywith 4 ml ice cold PBS+2% FCS. Subsequently, cells are centrifuged (5min 400 g), resuspended in 300-500 μL PBS and bound detection antibodyis quantified on a FACSCalibur or FACS Canto (BD (FL2 channel, 10.000cells per acquisition). During the experiment the respective isotypecontrols are included to exclude any unspecific binding events. Bindingof <IGF-1R> VL-VH/CL-CH1 exchange antibody and wildtype <IGF-1R>reference antibody to IGF-1R on 124 cells is compared via concentrationdependent shift of mean fluorescence intensity.

Additional experiments showed that <IGF-1R> VL-VH/CL-CH1 exchangeantibody also retained their activity to induce the internalization ofIGF-1R on MCF7 cells and had only low ADCC activity on DU145 cells ifincubated with human PBMCs comparable to the wildtype <IGF-1R> antibody

Taken together the experiments in example 1 showed that using the domainexchange fully functional antibodies with properties comparable towildtype antibodies can be generated. All functional properties wereretained in biochemical and cellular assays. These antibodies withdomain exchange form the basis for the generation of bispecificantibodies as described below.

Examples 2 Production, Expression, Purification and Characterization ofMonospecific, Bivalent <ANGPT2> Antibody, Wherein the Variable DomainsVL and VH are Replaced by Each Other, and Wherein the Constant DomainsCL and CH1 are Replaced by Each Other (Abbreviated Herein as <ANGPT2>VL-VH/CL-CH1 Exchange Antibody) Example 2A Making of the ExpressionPlasmids for the Monospecific, Bivalent <ANGPT2> VL-VH/CL-CH1 ExchangeAntibody Variant

The sequences for the heavy and light chain variable domains of amonospecific, bivalent Angiopoietin-2<ANGPT2> VL-VH/CL-CH1 exchangeantibody including the respective leader sequences described in thisexample are derived from a human <ANGPT2> antibody heavy chain (SEQ IDNO: 6) and a light chain (SEQ ID NO: 7) described in WO 2006/045049 andthe heavy and light chain constant domains are derived from a humanantibody (C-kappa and IgG1).

The gene segments encoding the <ANGPT2> antibody leader sequence, lightchain variable domain (VL) and the human kappa-light chain constantdomain (CL) were joined and fused to the 5′-end of the Fc domains of thehuman γ1-heavy chain constant domains (Hinge-CH2-CH3). The DNA codingfor the respective fusion protein resulting from the exchange of VH andCH1 domains by VL and CL domains was generated by gene synthesis and isdenoted <ANGPT2> HC* (heavy chain*) (SEQ ID NO: 8) in the following.

The gene segments for the <ANGPT2> antibody leader sequence, heavy chainvariable domain (VH) and the human γ1-heavy chain constant domains (CH1)were joined as independent chain. The DNA coding for the respectivefusion protein resulting from the exchange of VL and CL domains by VHand CH1 domains was generated by gene synthesis and is denoted <ANGPT2>LC* (light chain*) (SEQ ID NO: 9) in the following.

The respective expression vectors are analogous to the ones described inexample 1A. The plasmid map of the heavy chain* <ANGPT2> HC* expressionvector pUC-HC*-ANGPT2> is shown in FIG. 12. The amino acid sequence ofthe <ANGPT2> HC* (including signal sequence) is given in SEQ ID NO: 8.

The plasmid map of the light chain* <ANGPT2> LC* expression vectorpUC-LC*-ANGPT2> is shown in FIG. 13. The amino acid sequence of the<ANGPT2> LC* (including signal sequence) is given in SEQ ID NO: 9.Plasmids pUC-HC*-ANGPT2> and pUC-LC*-ANGPT2> can be used for transientor stable co-transfections e.g. into HEK293-F, HEK293 EBNA or CHO cells(2-vector system).

In order to achieve higher expression levels in transient expressions inHEK293 EBNA cells the <ANGPT2> HC* expression cassette can be sub-clonedvia AscI, SgrAI sites and the <ANGPT2> LC* expression cassette can besub-cloned via Sse8387I and FseI sites into the 4700 pUC-Hyg_OriPexpression vector as described in example 1A. Heavy and light chaintranscription units can either be sub-cloned into two independent4700-pUC-Hyg-OriP vectors for co-transfection (2-vector system) or theycan be cloned into one common 4700-pUC-Hyg-OriP vector (1-vector system)for subsequent transient or stable transfections with the resultingvectors via FseI, SgrAI, Sse8387I and AscI sites.

The wildtype <ANGPT2> antibody was cloned into plasmidsSB04-pUC-HC-ANGPT2 (SEQ ID NO: 6) and SB06-pUC-LC-ANGPT2 (SEQ ID NO: 7)that are analogous to the vectors described in this and previous example1A. The transcription units for the wildtype <ANGPT2> antibody heavy andlight chains were sub-cloned from plasmids SB04-pUC-HC-ANGPT2 andSB06-pUC-LC-ANGPT2 basic vectors via FseI, SgrAI, Sse8387I and AscIsites into plasmids SB07 -pUC-Hyg-OriP-HC-ANGPT2 andSB09-pUC-Hyg-OriP-LC-ANGPT2 in order to achieve higher transientexpression levels in HEK293E cells. For comparative reasons and forco-expression experiments (see example 3) the wildtype <ANGPT2> antibodywas either transiently (co-) expressed from plasmidsSB07-pUC-Hyg-OriP-HC-ANGPT2 and SB09-pUC-Hyg-OriP-LC-ANGPT2 or fromplasmids SB04-pUC-HC-ANGPT2 and SB06-pUC-LC-ANGPT2.

Example 2B Making of the Monospecific, Bivalent <ANGPT2> VL-VH/CL-CH1Exchange Antibody Expression Plasmids

The <ANGPT2> VL-VH/CL-CH1 exchange antibody fusion genes (HC* and LC*fusion genes) comprising the exchanged Fab sequences of the wildtype<ANGPT2> antibody were assembled with known recombinant methods andtechniques by connection of the according nucleic acid segments.

The nucleic acid sequences encoding the <ANGPT2> VL-VH/CL-CH1 exchangeantibody HC* and LC* were each synthesized by chemical synthesis andsubsequently cloned into a pPCRScript (Stratagene) based pGA4 cloningvector at Geneart (Regensburg, Germany). The expression cassetteencoding the <ANGPT2> VL-VH/CL-CH1 exchange antibody HC* was ligatedinto the respective E. coli plasmid via PstI and EcoNI restriction sitesresulting in the final vector pUC-HC*-<ANGPT2>; the expression cassetteencoding the respective <ANGPT2> LC* was ligated into the respective E.coli plasmid via PvuII and FseI restriction sites resulting in the finalvector pUC-LC*-<ANGPT2>. The subcloned nucleic acid sequences wasverified by DNA sequencing. For transient and stable transfectionslarger quantities of the plasmids were prepared by plasmid preparationfrom transformed E. coli cultures (Nucleobond AX, Macherey-Nagel)

Example 2C Transient Expression of Monospecific, Bivalent <ANGPT2>VL-VH/CL-CH1 Exchange Antibody in HEK293 EBNA Cells

Recombinant <ANGPT2> VL-VH/CL-CH1 exchange antibody was expressed bytransient co-transfection of plasmids pUC-HC*-ANGPT2> andpUC-LC*-ANGPT2> in adherently growing HEK293-EBNA cells (human embryonickidney cell line 293 expressing Epstein-Barr-Virus nuclear antigen;American type culture collection deposit number ATCC #CRL-10852, Lot.959 218) cultivated in DMEM (Dulbecco's modified Eagle's medium, Gibco)supplemented with 10% Ultra Low IgG FCS (fetal calf serum, Gibco), 2 mML-Glutamine (Gibco), and 250 μg/ml Geneticin (Gibco). For transfectionFuGENE™ 6 Transfection Reagent (Roche Molecular Biochemicals) was usedin a ratio of FuGENE™ reagent (μl) to DNA (μg) of 4:1 (ranging from 3:1to 6:1). Light and heavy chain plasmids encoding <ANGPT2> HC* and LC*(plasmids pUC-HC*-ANGPT2> and pUC-LC*-ANGPT2>) were expressed from twodifferent plasmids using a molar ratio of light chain to heavy chainencoding plasmids of 1:1 (equimolar) ranging from 1:2 to 2:1,respectively. Cells were feeded at day 3 with L-Glutamine ad 4 mM,Glucose [Sigma] and NAA [Gibco]. <ANGPT2> VL-VH/CL-CH1 exchange antibodycontaining cell culture supernatants were harvested from day 5 to 11after transfection by centrifugation and stored at −20° C. Generalinformation regarding the recombinant expression of humanimmunoglobulins in e.g. HEK293 cells is given in: Meissner, P. et al.,Biotechnol. Bioeng. 75 (2001) 197-203.

Example 2D Purification of Monospecific, Bivalent <ANGPT2> VL-VH/CL-CH1Exchange Antibody and Confirmation of Identity by Mass Spectrometry

The expressed and secreted monospecific, bivalent <ANGPT2> VL-VH/CL-CH1exchange antibody was purified from filtered cell culture supernatantsby Protein A affinity chromatography according to known standardmethods. In brief, the <ANGPT2> VL-VH/CL-CH1 exchange antibodycontaining cell culture supernatants from transient transfections wereclarified by centrifugation (10,000 g for 10 minutes) and filtrationthrough a 0.45 μm filter and applied to a Protein A HiTrap MabSelectXtra column (GE Healthcare) equilibrated with PBS buffer (10 mM Na2HPO4,1 mM KH2PO4, 137 mM NaCl and 2.7 mM KCl, pH 7.4). Unbound proteins werewashed out with PBS equilibration buffer followed by 0.1 M sodiumcitrate buffer, pH 5.5 and washed with PBS. Elution of VL-VH/CL-CH1exchange antibody was achieved with 100 mM sodium citrate, pH 2.8followed by immediate neutralization of the sample with 300 μl M Tris pH9.0 per 2 ml fraction. Aggregated protein was separated from monomericantibodies by size exclusion chromatography on a HiLoad 26/60 Superdex200 prep grade column (GE Healthcare) in 20 mM Histidine, 150 mM NaCl pH6.0 and monomeric antibody fractions were subsequently concentratedusing a MILLIPORE Amicon Ultra-15 centrifugal concentrator. <ANGPT2>VL-VH/CL-CH1 exchange antibody was frozen and stored at −20° C. or −80°C. The integrity of the <ANGPT2> VL-VH/CL-CH1 exchange antibody wasanalyzed by SDS-PAGE in the presence and absence of a reducing agent andsubsequent staining with Coomassie brilliant blue (FIG. 23-A).Aggregation and oligomeric state of the <ANGPT2> VL-VH/CL-CH1 exchangeantibody was analyzed by analytical size exclusion chromatography (FIG.23-B) Characterized samples were provided for subsequent proteinanalytics and functional characterization. ESI mass spectrometryconfirmed the theoretical molecular mass of the completelydeglycosylated <ANGPT2> VL-VH/CL-CH1 exchange antibody as main species.No contamination by antibody-like proteins could be observed.

Example 2F Analysis of the <ANGPT2> Binding Properties of Monospecific,Bivalent <ANGPT2> VL-VH/CL-CH1 Exchange Antibody in an <ANGPT2> BindingELISA and by Biacore

The binding properties of monospecific, bivalent <ANGPT2> VL-VH/CL-CH1exchange antibody were evaluated in an ELISA assay with full-lengthANGPT2>-His protein (R&D Systems) as described above. FIG. 24 shows theresults from a titration of purified <IGF-1R> VL-VH/CL-CH1 exchangeantibody and wildtype <IGF-1R> antibody in the sandwich binding ELISA.It can be clearly seen that <ANGPT> VL-VH/CL-CH1 exchange antibody isfunctional and shows comparable binding properties as the wildtype<ANGPT2> antibody and thus appears fully functional. The minor observeddifferences lie within the error of the method and might e.g. resultfrom minor variances in protein concentration.

These findings were corroborated by Biacore data with the respectivepurified antibodies that showed that monospecific, bivalent <ANGPT2>VL-VH/CL-CH1 exchange antibody with a KD value of 13 pM had a comparableaffinity and binding kinetics for ANGPT2 within the error of the methodas the original wildtype <ANGPT2> antibody with a KD value of 12 pM(FIG. 25)

Example 2G Analysis of the Functional Properties of Monospecific,Bivalent <ANGPT2> VL-VH/CL-CH1 Exchange Antibody

In order to show that the monospecific, bivalent <ANGPT2> VL-VH/CL-CH1exchange antibody has functional properties comparable to the originalwildtype <ANGPT2> antibody the two antibodies were compared for theirability to interfere with binding of ANGPT2 to its Tie2 receptorextracellular domain in an ELISA binding assay as described above. Inthe respective binding ELISA <ANGPT2> VL-VH/CL-CH1 exchange antibody hadan EC50 value of 135 ng/ml for interference with binding of human ANGPT2which is comparable to the EC50 value of 145 nM for the originalwildtype <ANGPT2> antibody. These data were confirmed in a cellularligand binding competition assay with HEK293 cells over-expressing Tie2on their surface where <ANGPT2> VL-VH/CL-CH1 exchange antibody had anEC50 value of 225 ng/ml for interference with binding of human ANGPT2which is comparable to the EC50 value of 205 nM for the originalwildtype <ANGPT2> antibody (data not shown).

Taken together the experiments in example 2 showed that using the domainexchange fully functional antibodies with properties comparable towildtype antibodies can be generated. All functional properties wereretained in biochemical and cellular assays. These antibodies withdomain exchange form the basis for the generation of bispecificantibodies as described below in examples 3 and 4.

Example 2H

A molecular modeling analysis of the antibodies with domain exchangerevealed that the steric room between the exchanged domains could belimiting folding. Therefore constructs were designed in which anadditional Glycine residue was introduced at the C-terminus of theexchanged domains of the Fab e.g. between the VL-CL and Hinge connectingregion or at the end of free VH-CH1 domain respectively. The respectivemonospecific, bivalent antibody is denoted as <ANGPT2>VL-VH(G)/CL-CH1(G) exchange antibody in the following.

The sequences for the heavy and light chain variable domains of amonospecific, bivalent Angiopoietin-2<ANGPT2> VL-VH/CL-CH1 exchangeantibody including the respective leader sequences described in thisexample are derived from a human <ANGPT2> antibody heavy chain (SEQ IDNO: 6) and a light chain (SEQ ID NO: 7) described in WO 2006/045049 andthe heavy and light chain constant domains are derived from a humanantibody (C-kappa and IgG1).

The gene segments encoding the <ANGPT2> antibody leader sequence, lightchain variable domain (VL) and the human kappa-light chain constantdomain (CL) were joined and fused to the 5′-end of the Fc domains of thehuman γ1-heavy chain constant domains (Hinge-CH2-CH3) and including anadditional Glycine residue. The DNA coding for the respective fusionprotein resulting from the exchange of VH and CH1 domains by VL and CLdomains was generated by gene synthesis and is denoted <ANGPT2> HC(G)*(heavy chain*) (SEQ ID NO: 17) in the following.

The gene segments for the <ANGPT2> antibody leader sequence, heavy chainvariable domain (VH) and the human γ1-heavy chain constant domains (CH1)were joined as independent chain including an additional Glycineresidue. The DNA coding for the respective fusion protein resulting fromthe exchange of VL and CL domains by VH and CH1 domains was generated bygene synthesis and is denoted <ANGPT2> LC(G)* (light chain*) (SEQ ID NO:18) in the following.

The respective expression vectors are analogous to the ones described inexamples 2 above. Subsequently, these expression vectors were used forthe transient co-expression of the respective <ANGPT2>VL-VH(G)/CL-CH1(G) antibody with domain exchange in HEK293-F cells. Therespective <ANGPT2> VL-VH(G)/CL-CH1(G) exchange antibody was purifiedfrom transient expression as described above and analyzed by SDS-PAGE,size exclusion chromatography and mass spectrometry of the respectiveantibody. Protein expression yields were good and similar to theexpression yields obtained for the conventional VL-VH/CL-CH1 exchangeantibody described above. All properties investigated showed nounexpected findings and were essentially comparable to the respective<ANGPT2 wildtype antibody. FIG. 31 demonstrates the virtually comparableproperties of the <ANGPT2> wildtype antibody and the <ANGPT2>VL-VH(G)/CL-CH1(G) antibody in an ANGPT2 Biacore binding assay. Withinthe error of the method both antibodies exhibited a comparable bindingaffinity for human ANGPT2 with estimated KD values (mean from twodeterminations) of ca. 38 pM for the <ANGPT2> wildtype antibody and ca.45 pM for the <ANGPT2> VL-VH(G)/CL-CH1(G) antibody.

[M] ka [1/Ms] kd [1/s] K(D) wildtype <ANGPT2> antibody 5.26E+05  2.0E−43.79E−10 <ANGPT2> VL-VH(G)/CL-CH1(G) 5.29E+06 2.358E−3 4.45E−09 exchangeantibody

Examples 3 Expression of Bispecific, Bivalent <ANGPT2-IGF-1R> Antibody,Wherein in the Heavy and Light Chain Specifically Binding to IGF-1R, theVariable Domains VL and VH are Replaced by Each Other, and the ConstantDomains CL and CH1 are Replaced by Each Other (Abbreviated Herein as<ANGPT2-IGF-1R> VL-VH/CL-CH1 Exchange Antibody) Example 3A

Transient co-expression of <ANGPT2> wildtype antibody and <IGF-1R>VL-VH/CL-CH1 exchange antibody in HEK293 EBNA cells to yield bispecific<ANGPT2-IGF-1R> VL-VH/CL-CH1 exchange antibody

In order to generate a functional bispecific antibody recognizing<ANGPT2> via the <ANGPT2> wildtype Fab region on one side and IGF-1R viathe <IGF-1R> VL-VH/CL-CH1 exchange antibody Fab region on the other sidethe two expression plasmids coding for the <IGF-1R> VL-VH/CL-CH1exchange antibody (example 1A) were co-expressed with two expressionplasmids coding for the <ANGPT2> wildtype antibody. (example 2A).Assuming a statistical association of wildtype heavy chains HC and Fabcrossover heavy chains HC* this resulted in the generation of bispecificand bivalent <ANGPT2-IGF-1R> VL-VH/CL-CH1 exchange antibody. Under theassumption that both antibodies are equally well expressed and withouttaking side products into account this should have resulted in a 1:2:1ratio of the three main products monospecific <IGF-1R> VL-VH/CL-CH1exchange antibody, bispecific <ANGPT2-IGF-1R> VL-VH/CL-CH1 exchangeantibody, and <ANGPT2> wildtype antibody. In addition, several sideproducts such as LC-LC* (Fab2 fragment), HC-HC* (monovalent antibody)and HC*-LC dimers can be expected.

In contrast, when co-expressing the two expression plasmids coding forthe <IGF-1R> wildtype antibody (example 1A) and the two expressionplasmids coding for the <ANGPT2> wildtype antibody as a reference only asmall proportion of functional bispecific <IGF-1R-ANGPT2> antibody willbe generated due to the statistical association of the heavy chains butthe unguided association of light chains with both heavy chains from the<IGF-1R> and <ANGPT2> wildtype antibodies.

To generate the mix of the main products A) monospecific <IGF-1R>VL-VH/CL-CH1 exchange antibody, B) bispecific <ANGPT2-IGF-1R>VL-VH/CL-CH1 exchange antibody and C) <ANGPT2> wildtype antibodies thefour plasmids DW047-pUC-HC*-IGF-1R and DW048-pUC-LC*-IGF-1R and eitherplasmids SB07-pUC-Hyg-OriP-HC-ANGPT2 and SB09-pUC-Hyg-OriP-LC-ANGPT2 (orplasmids SB04-pUC-HC-ANGPT2 and SB06-pUC-LC-ANGPT2) were transientlyco-transfected in adherently growing HEK293-EBNA cells (human embryonickidney cell line 293 expressing Epstein-Barr-Virus nuclear antigen;American type culture collection deposit number ATCC #CRL-10852, Lot.959 218) cultivated in DMEM (Dulbecco's modified Eagle's medium, Gibco)supplemented with 10% Ultra Low IgG FCS (fetal calf serum, Gibco), 2 mML-Glutamine (Gibco), and 250 μg/ml Geneticin (Gibco). For transfectionFuGENE™ 6 Transfection Reagent (Roche Molecular Biochemicals) was usedin a ratio of FuGENE™ reagent (μl) to DNA (μg) of 4:1 (ranging from 3:1to 6:1). Light and heavy chain plasmids encoding <IGF-1R> HC* and LC*(plasmids DW047-pUC-HC*-IGF-1R and DW048-pUC-LC*-IGF-1R) and <ANGPT2> HCand LC (plasmids SB07-pUC-Hyg-OriP-HC-ANGPT2 andSB09-pUC-Hyg-OriP-LC-ANGPT2 or plasmids SB04-pUC-HC-ANGPT2 andSB06-pUC-LC-ANGPT2, respectively) were expressed from four differentplasmids using a molar ratio of light chains to heavy chains encodingplasmids of 1:1 (equimolar). Cells were feeded at day 3 with L-Glutaminead 4 mM, Glucose [Sigma] and NAA [Gibco]. The harvested supernatantcontained a mix of the main products A) monospecific <IGF-1R>VL-VH/CL-CH1 exchange antibody, B) bispecific <ANGPT2-IGF-1R>VL-VH/CL-CH1 exchange antibody and C)<ANGPT2> wildtype antibodies and isdenoted as “Bispecific VL-VH/CL-CH1 exchange mix”. BispecificVL-VH/CL-CH1 exchange mix containing cell culture supernatants, wereharvested from day 5 to 11 after transfection by centrifugation andstored at −20° C.

For comparative reasons the wildtype <IGF-1R> antibody was transientlyco-expressed from HC and LC plasmids (4842-pUC-LC-IGF-1R and4843-pUC-HC-IGF-1R, example 1A) together with the wildtype <ANGPT2> HCand LC plasmids (SB07-pUC-Hyg-OriP-HC-ANGPT2 andSB09-pUC-Hyg-OriP-LC-ANGPT2 or SB04-pUC-HC-ANGPT2 andSB06-pUC-LC-ANGPT2, respectively). Cells were feeded at day 3 withL-Glutamine ad 4 mM, Glucose [Sigma] and NAA [Gibco]. The harvestedsupernatant contained a mix of different <IGF-1R> and <ANGPT2> wildtypeantibody variants either monospecific, bispecific or binding incompetentdue to the unguided association of light chains with both heavy chainsfrom the <IGF-1R> and <ANGPT2> wildtype antibodies resulting in wronglight chain association, and is denoted as “wildtype mix”. Wildtype mixcontaining cell culture supernatants, were harvested from day 5 to 11after transfection by centrifugation and stored at −20° C.

As the <ANGPT2> wildtype antibody shows significantly higher expressiontransient expression yields than the <IGF-1R> wildtype and <IGF-1R> Fabcrossover antibodies the ratio of <ANGPT2> wildtype antibody plasmidsand <IGF-1R> VL-VH/CL-CH1 exchange antibody plasmids was shifted infavour of the expression of <ANGPT2> wildtype antibody. The plasmidratio has to be adapted during future experiments to allow an equalexpression of both specificities resulting in a more even distributionof the different antibodies.

Example 3B Immunoprecipitation of Bispecific <ANGPT2-IGF-1R>VL-VH/CL-CH1 Exchange Antibody

A mix of the main products A) monospecific <IGF-1R> VL-VH/CL-CH1exchange antibody, B) bispecific <ANGPT2-IGF-1R> VL-VH/CL-CH1 exchangeantibody and C) <ANGPT2> wildtype antibodies (“Bispecific VL-VH/CL-CH1exchange mix”) was isolated from cell culture supernatants (example 3A)by immunoprecipitation with Protein A Agarose-beads (Roche). 60 μLProtein A Agarose beads were washed three times in TBS-NP40 (50 mM Tris,pH 7.5, 150 mM NaCl, 1% Nonidet-P40). Subsequently, 1-15 mL cell culturesupernatant were applied to the Protein A Agarose beads pre-equilibratedin TBS-NP40. After incubation for at 1 h at room temperature the beadswere washed on an Ultrafree-MC-filter column (Amicon] once with 0.5 mLTBS-NP40, twice with 0.5 mL 2× phosphate buffered saline (2×PBS, Roche)and briefly four times with 0.5 mL 100 mM Na-citrate pH 5.0. Boundantibody was eluted by addition of 35 μl NuPAGE® LDS Sample Buffer(Invitrogen). Half of the sample was combined with NuPAGE® SampleReducing Agent or left unreduced, respectively, and heated for 10 min at70° C. Consequently, 20 μl were applied to an 4-12% NuPAGE® Bis-TrisSDS-PAGE (Invitrogen) (with MOPS buffer for non-reduced SDS-PAGE and MESbuffer with NuPAGE® Antioxidant running buffer additive (Invitrogen) forreduced SDS-PAGE) and stained with Coomassie Blue. On the SDS-PAGE nodifference between the three antibody species could be seen. Allantibodies behaved like typical IgG1 antibodies with a ca. 25 kDa bandcorresponding to the light chains and a 50 kDa band corresponding to therespective heavy chains. In the unreduced state, a band at ca. 150 kDacould be observed for the complete antibody. Thus, in order to prove thepresence of functional bispecific <ANGPT2-IGF-1R> VL-VH/CL-CH1 exchangeantibody the crossover mix of the three antibody species was purified(example 3C) and a cellular FACS bridging assay was designed (example3D).

Example 3C Purification of Bispecific <ANGPT2-IGF-1R> VL-VH/CL-CH1Exchange Antibody

The mix of the main products A) monospecific <IGF-1R> VL-VH/CL-CH1exchange antibody, B) bispecific <ANGPT2-IGF-1R> VL-VH/CL-CH1 exchangeantibody and C) <ANGPT2> wildtype antibodies (“Bispecific VL-VH/CL-CH1exchange mix”) from example 3A was purified from filtered cell culturesupernatants by Protein A affinity chromatography followed by sizeexclusion chromatography according to known standard methods. In brief,the antibody mix containing cell culture supernatants from transienttransfections of plasmids DW047-pUC-HC*-IGF-1R and DW048-pUC-LC*-IGF-1Rand SB07-pUC-Hyg-OriP-HC-ANGPT2 and SB09-pUC-Hyg-OriP-LC-ANGPT2 wereclarified by centrifugation (10,000 g for 10 minutes) and filtrationthrough a 0.45 μm filter and applied to a Protein A HiTrap MabSelectXtra column (GE Healthcare) equilibrated with PBS buffer (10 mM Na2HPO4,1 mM KH2PO4, 137 mM NaCl and 2.7 mM KCl, pH 7.4). Unbound proteins werewashed out with PBS equilibration buffer followed by 0.1 M sodiumcitrate buffer, pH 5.5 and washed with PBS. Elution of bispecific<ANGPT2-IGF-1R> VL-VH/CL-CH1 exchange antibody was achieved with 100 mMsodium citrate, pH 2.8 followed by immediate neutralization of thesample with 300 μl M Tris pH 9.0 per 2 ml fraction. Aggregated proteinwas separated from monomeric antibodies by size exclusion chromatographyon a HiLoad 26/60 Superdex 200 prep grade column (GE Healthcare) in 20mM Histidine, 150 mM NaCl pH 6.0 and monomeric antibody fractions weresubsequently concentrated using a MILLIPORE Amicon Ultra-15 centrifugalconcentrator. The VL-VH/CL-CH1 exchange mix was frozen and stored at−20° C. or −80° C. The integrity of the antibody species was analyzed bySDS-PAGE (see FIG. 14) in the presence and absence of a reducing agentand subsequent staining with Coomassie brilliant blue as described inExample 1D. The antibody mix behaved like typical IgG1 antibodies with aca. 25 kDa band corresponding to the light chains and a 50 kDa bandcorresponding to the respective heavy chains. In the unreduced state aband at ca. 150 kDa could be observed for the complete antibody. Noobvious side-products such as monovalent antibodies etc. were visiblefrom the SDS-PAGE after Protein A purification. Aggregation andoligomeric state of the bispecific <ANGPT2-IGF-1R> VL-VH/CL-CH1 exchangeantibody was analyzed by analytical size exclusion chromatography andshowed that the purified antibody species were in a monomeric state.Characterized samples were provided for subsequent protein analytics andfunctional characterization. In order to prove the presence offunctional bispecific <ANGPT2-IGF-1R> VL-VH/CL-CH1 exchange antibody themix of the three antibody species was analyzed in a cellular FACSbridging assay (example 3D).

For comparative reasons the wildtype mix resulting from theco-expression of wildtype <IGF-1R> antibody HC and LC plasmids(4842-pUC-LC-IGF-1R and 4843-pUC-HC-IGF-1R, example 1A) together withthe wildtype <ANGPT2> HC and LC plasmids (SB07-pUC-Hyg-OriP-HC-ANGPT2and SB09-pUC-Hyg-OriP-LC-ANGPT2) was purified as a reference fromfiltered cell culture supernatants by Protein A affinity chromatographyfollowed by size exclusion chromatography according to the describedprocedure.

Example 3D Detection of Functional Bispecific <ANGPT2-IGF-1R>VL-VH/CL-CH1 Exchange Antibody in a Cellular FACS Bridging Assay on 124IGF-1R Expressing Cells

In order to confirm the presence of functional bispecific<ANGPT2-IGF-1R> VL-VH/CL-CH1 exchange antibody in the “BispecificVL-VH/CL-CH1 exchange mix” of the main products A) monospecific <IGF-1R>VL-VH/CL-CH1 exchange antibody, B) bispecific <ANGPT2-IGF-1R>VL-VH/CL-CH1 exchange antibody and C)<ANGPT2> wildtype antibodies fromthe transient co-expression described in example 3A, a cellular FACSIGF-1R-ANGPT2 bridging assay on I24 cells (NIH3T3 cells expressingrecombinant human IGF-1R, Roche) was performed. The assay principle isdepicted in FIG. 15. A bispecific <ANGPT2-IGF-1R> VL-VH/CL-CH1 exchangeantibody that is present in the supernatant (example 3A) or in thepurified antibody mix (example 3C), respectively; is capable of bindingto IGF-1R in 124 cells and to ANGPT2 simultaneously; and thus willbridge its two target antigens with the two opposed Fab regions.

Briefly, 5×10E5 I24 cells per FACS tube were incubated with either 50 μLundiluted cell culture supernatant or with a 160 μg/mL dilution of totalpurified antibody mix and incubated on ice for 1 h. In the first casethe cells were incubated with A) cell culture supernatants from theco-expression of the crossover <IGF-1R> plasmids (DW047-pUC-HC*-IGF-1Rand DW048-pUC-LC*-IGF-1R) with the wildtype <ANGPT2> plasmids(SB04-pUC-HC-ANGPT2> and SB06-pUC-LC-ANGPT2) denoted as “BispecificVL-VH/CL-CH1 exchange mix” or B) with cell culture supernatant from theco-expression of the wildtype <IGF-1R> plasmids (4842-pUC-LC-IGF-1R and4843-pUC-HC-IGF-1R) with the wildtype <ANGPT2> plasmids(SB04-pUC-HC-ANGPT2> and SB06-pUC-LC-ANGPT2) denoted as “Wildtype mix”(FIG. 16). In the second case, the respective purified antibody mix fromeither Bispecific VL-VH/CL-CH1 exchange mix or from Wildtype mix wasapplied to the 124 cells (example 3C, FIG. 17). Unbound antibody waswashed away with 4 ml ice cold PBS (Gibco)+2% FCS (Gibco), cells werecentrifuged (5 min at 400 g) and bound bispecific antibody was detectedwith 50 μl 2 μg/mL human Angiopoietin-2 (ANGPT2) (R&D Systems) for 1 hon ice. Subsequently, unbound Angiopoietin-2 (ANGPT2) was washed awayonce (FIG. 16) or twice (FIG. 17) with 4 ml ice cold PBS (Gibco)+2% FCS(Gibco), cells were centrifuged (5 min at 400 g) and boundAngiopoietin-2 was detected with 50 μl 5 μg/mL <Ang-2> mIgG1-Biotinantibody (BAM0981, R&D Systems) for 45 min on ice; alternatively, cellswere incubated with 50 μl 5 μg/mL mIgG1-Biotin-Isotype control (R&DSystems). Unbound detection antibody was washed away with 4 ml ice coldPBS (Gibco)+2% FCS (Gibco), cells were centrifuged (5 min at 400 g) andbound detection antibody was detected with 50 μl 1:400 Streptavidin-PEconjugate (Invitrogen/Zymed) for 45 min on ice protected from light.Unbound Streptavidin-PE conjugate was washed away with 4 ml ice coldPBS+2% FCS. Subsequently, cells were centrifuged (5 min 400 g),resuspended in 300-500 μL PBS and bound Streptavidin-PE conjugate wasquantified on a FACSCalibur or FACS Canto (BD (FL2 channel, 10.000 cellsper acquisition). During the experiment the respective isotype controlswere included to exclude any unspecific binding events. In addition,purified monospecific, bivalent IgG1 antibodies <IGF-1R> and <ANGPT2>were included as controls.

FIG. 16 shows the results from the cellular FACS bridging assay on 124cells with cell culture supernatants; FIG. 17 shows the results with thepurified antibody mix. In both cases in which the VL-VH/CL-CH1 exchangetechnology was applied, the incubation with supernatant or purifiedantibody “Bispecific VL-VH/CL-CH1 exchange mix” from the co-expressionof a VL-VH/CL-CH1 exchange antibody with a wildtype antibody resulted ina significant shift in fluorescence indicating the presence of afunctional bispecific <ANGPT2-IGF-1R> VL-VH/CL-CH1 exchange antibodythat is capable of binding to IGF-1R in 124 cells and to ANGPT2simultaneously; and thus bridges its two target antigens with the twoopposed Fab regions. In contrast to this and as predicted for theco-expression of two wildtype antibodies, only a very small proportionof functional bispecific antibodies is formed resulting in only a slightshift in fluorescence in the FACS bridging assay when the cell culturesupernatant or purified antibody “Wildtype mix” from the co-expressionof the wildtype <IGF-1R> plasmids with the wildtype <ANGPT2> plasmidswas applied indicating the presence of only a small fraction offunctional bispecific <IGF-1R-ANGPT2> wildtype antibody.

These results show that by co-expression of VL-VH/CL-CH1 exchangeplasmids coding for a <IGF-1R> antibody with wildtype plasmids codingfor a <ANGPT2> antibody functional bispecific <ANGPT2-IGF-1R>VL-VH/CL-CH1 exchange antibody recognizing the two different targetssimultaneously can easily be generated. In contrast, the co-expressionof two wildtype plasmids coding for <IGF-1R> and <ANGPT2> antibodiesresults in a high complexity of the formed antibodies and only a minorproportion of functional bispecific <IGF-1R-ANGPT2> wildtype antibody.

Example 4 Generation of Bivalent, Bispecific <VEGF-ANGPT2> VL-VH/CL-CH1Exchange Antibody with Modified CH3 Domains (Knobs-into-Holes) (FIG. 30)

As shown in the example above the co-expression of plasmids coding forwildtype antibodies with plasmids coding for VL-VH/CL-CH1 exchangeantibodies results in the generation of a bispecific VL-VH/CL-CH1exchange mix of the main products A) monospecific VL-VH/CL-CH1 exchangeantibody, B) bispecific VL-VH/CL-CH1 exchange antibody and C) wildtypeantibody. To further improve the yield of the bispecific <VEGF-ANGPT2>VL-VH/CL-CH1 exchange antibody the knobs-into-holes technology wasapplied to the co-expression of a <ANGPT2> VL-VH/CL-CH1 exchangeantibody and a wildtype <VEGF> antibody to foster heterodimerization ofthe respective unmodified and modified heavy chains (via HC and HC*) andobtain a more homogenous and functional bispecific antibody preparation.In this example a bispecific antibody recognizing VEGF and ANGPT2simultaneously based on the <VEGF> wildtype antibody G6-31 and theVL-VH/CL-CH1 exchange <ANGPT2> antibody Mab536 as described above wasgenerated.

As described above the gene segments encoding the <ANGPT2> antibodyleader sequence, light chain variable domain (VL) and the humankappa-light chain constant domain (CL) were joined and fused to the5′-end of the Fc domains of the human γ1-heavy chain constant domains(Hinge-CH2-CH3). The DNA coding for the respective fusion proteinresulting from the exchange of VH and CH1 domains by VL and CL domainswas generated by gene synthesis and is denoted <ANGPT2> HC* (heavychain*) (SEQ ID NO: 8).

The gene segments for the <ANGPT2> antibody leader sequence, heavy chainvariable domain (VH) and the human g1-heavy chain constant domains (CH1)were joined as independent chain. The DNA coding for the respectivefusion protein resulting from the exchange of VL and CL domains by VHand CH1 domains was generated by gene synthesis and is denoted <ANGPT2>LC* (light chain*) (SEQ ID NO: 9) in the following.

The sequences for the wildtype heavy and light chain variable domains ofthe monospecific, bivalent <VEGF> wildtype antibody G6-31 including therespective leader sequences described in this example are derived fromthe human <VEGF> antibody G6-31 heavy chain variable domains of SEQ IDNO: 12 and the light chain variable domains of SEQ ID NO: 13 which areboth derived from the human phage display derived anti-VEGF antibodyG6-31 which is described in detail in Liang et al., J Biol. Chem. 2006Jan. 13; 281(2):951-61 and in US 2007/0141065 and the heavy and lightchain constant domains are derived from a human antibody (C-kappa andIgG1).

For this purpose, the plasmid coding for the for the heavy chain of the<VEGF> wildtype antibody G6-31 was modified by introduction of a CH3gene segment generated by gene synthesis and coding for the knobsresidue T366W of the SEQ ID NO: 10 into the CH3 domain of the respective<VEGF> wildtype antibody G6-31 heavy chain resulting in SEQ ID NO: 16;in contrast the plasmid coding for the heavy chain* HC* of theVL-VH/CL-CH1 exchange <ANGPT2> antibody Mab536 was modified byintroduction of a CH3 gene segment generated by gene synthesis andcoding for the hole residues T366S, L368A, Y407V of the SEQ ID NO: 11into the CH3 domain of the respective VL-VH/CL-CH1 exchange <ANGPT2>antibody Mab536 heavy chain* HC* resulting in SEQ ID NO: 15. In thiscase plasmids coding for the wildtype <VEGF> knobs antibody G6-31 wereorganized as genomic vectors whereas the plasmids for the VL-VH/CL-CH1exchange <ANGPT2> antibody Mab536 were organized as IntronA-cDNAvectors.

Subsequently the respective four plasmids coding for <VEGF> G6-31 heavychain HC with knob SEQ ID NO: 16 and wildtype light chain LC SEQ ID NO:14, and the modified VL-VH/CL-CH1 exchange <ANGPT> heavy chain* HC* withhole SEQ ID NO: 15 and modified light chain* LC* (SEQ ID NO: 8) wereco-expressed in an equimolar ratio by transient transfections in theHEK293-F system as described above in a 2.6 L scale in a Quad fermenter.Antibodies were purified via a HiTrap MabSelect column (GE) followed bysize exclusion chromatography on a HiLoad Superdex 200 26/60 column (GE)as described above. Yields after SEC were in the range of ca. 38 mgpurified antibody fractions from 2 L transient expression. FIG. 26 showsa reduced and non-reduced SDS-PAGE of the size exclusion fractionsobtained during the purification. On the reduced SDS-PAGE it can be seenthat the respective wildtype and modified heavy and light chains havebeen synthesized and purified homogeneously. However, on the non-reducedSDS-PAGE it is obvious that there were still several antibody speciespresent.

In the following the obtained size exclusion fractions were analyzed bymass spectrometry (either deglycosylated, but not reduced ordeglycosylated and reduced). Several antibody species could beidentified in the fractions by mass spectrometry and could also beassigned to the bands that are seen on the respective SDS-PAGE (FIG.27). The following table shows the antibody species that could beidentified:

1. Run 1. Run Frac 5 Frac 9 Species deglyc. deglyc. LC G6-31/23255Da/(SS): 23251 Da — — HC G6-31/49149 Da/(SS): 49136 Da — — LC*VL-VH/CL-CH1 exchange <ANGPT2> — — antibody/23919 Da/(SS): 23914 Da HC*VL-VH/CL-CH1 exchange <ANGPT2> — antibody Mab536/49245 Da/(SS): 49231 DaLC G6-31 + LC VL-VH/CL-CH1 exchange X X <ANGPT2> antibody Mab536 =>(SS): 47165 Da Complete Ab G6-31(SS) 144774 Da X Complete Ab bispecific<VEGF-ANGPT2> X VL-VH/CL-CH1 exchange antibody (SS) 145532 Da (LC + HCG6-31) + (LC* + HC*) ½ Ab G6-31 (SS) 72387 Da X X (HC G6-31) + (HCVL-VH/CL-CH1 X X exchange <ANGPT2> antibody Mab536)=> (SS) 98367 Da (SH)2 × (HC VL-VH/CL-CH1 exchange X <ANGPT2> antibody Mab536) + LC G6-31 =>121745 Da 2 × (HC G6-31) + LC G6-31 + HC VL- X + 20 Da VH/CL-CH1exchange <ANGPT2> antibody Mab536 =>(SS): 170754 Da

The desired bispecific <VEGF-ANGPT2> VL-VH/CL-CH1 exchange antibodyrecognizing VEGF with one arm and ANGPT2 with the other arm could thusbe unequivocally be identified by mass spectrometry and could also beseen on the respective SDS-PAGE. A schematic scheme of the desiredbispecific <VEGF-ANGPT2> VL-VH/CL-CH1 exchange antibody is seen in FIG.30. The highest concentration of this desired antibody could be found infractions 5 and 6 whereas its concentration was reduced in latterfractions.

In addition, the presence of bispecific <VEGF-ANGPT2> VL-VH/CL-CH1exchange antibody capable of binding simultaneously to ANGPT2 and VEGFwas confirmed by an ELISA bridging assay (FIG. 28) and a Biacorebridging assays (FIG. 29) described above showing that the desiredbispecific antibody was capable of binding to ANGPT2 and VEGFsimultaneously. In these assays a tetravalent bispecific antibodyTvG6-Ang23 served as a control.

It should be noted that due to a relative over-expression of thewildtype <VEGF> antibody G6-31 e.g. due to better expression yields forthe genomic vectors coding for G6-31 derivatives the formation ofinactive side products was fostered resulting in an excess of inactiveG6-31 dimers and half antibodies. In order to achieve a maximal yield ofthe desired bispecific <VEGF-ANGPT2> VL-VH/CL-CH1 exchange antibodyadditional studies are ongoing that achieve a uniform expression of allfour antibody chains and reduce side product formation. These studiesinclude i) the optimization of the equimolar stochiometry of the 4plasmids used for o-expression e.g. by combining the transcriptionalunits on one or two plasmids, the introduction of the respective controlelements; as well ii) the optimization of heterodimerization e.g. byusing different knobs-in-holes technologies such as the introduction ofan additional disulfide bridge into the CH3 domain e.g. Y349C into the“knobs chain” and D356C into the “hole chain” and/or combined with theuse of residues R409D; K370E (K409D) for knobs residues and D399K; E357Kfor hole residues described by EP 1870459A1.

The invention claimed is:
 1. A bivalent, bispecific antibody,comprising: a) the light chain and heavy chain of an antibodyspecifically binding to a first antigen; and b) the light chain andheavy chain of an antibody specifically binding to a second antigen,wherein: within the light chain of the antibody which binds to saidsecond antigen, the variable light chain domain VL is replaced by thevariable heavy chain domain VH of said antibody and the constant lightchain domain CL is replaced by the constant heavy chain domain CH1 ofsaid antibody; and, within the heavy chain of the antibody which bindsto said second antigen, the variable heavy chain domain VH is replacedby the variable light chain domain VL of said antibody, and the constantheavy chain domain CH1 is replaced by the constant light chain domain CLof said antibody.
 2. The antibody according to claim 1, wherein the CH3domain of one heavy chain and the CH3 domain of the other heavy chaineach meet at an interface: wherein the CH3 domain of one heavy chain isaltered, so that within the interface the CH3 domain of one heavy chainthat meets the interface of the CH3 domain of the other heavy chainwithin the bivalent, bispecific antibody, one or two amino acid residuesare replaced with an equivalent number of amino acid residues having alarger side chain volume, thereby generating a protuberance within theinterface of the CH3 domain of one heavy chain which is positionable ina cavity within the interface of the CH3 domain of the other heavy chainand wherein the CH3 domain of the other heavy chain is altered so thatwithin the interface of the second CH3 domain that meets the interfaceof the first CH3 domain within the bivalent, bispecific antibody two orthree amino acid residues are replaced with an equivalent number ofamino acid residues having a smaller side chain volume, therebygenerating a cavity within the interface of the second CH3 domain withinwhich a protuberance within the interface of the first CH3 domain ispositionable.
 3. The antibody according to claim 2, wherein one aminoacid residue is replaced with an amino acid having a larger side chainvolume, and wherein the amino acid residue having a larger side chainvolume is selected from the group consisting of arginine (R),phenylalanine (F), tyrosine (Y), and tryptophan (W).
 4. The antibodyaccording to claim 2, wherein three amino acid residues are replacedwith three amino residues having a smaller side chain volume, whereinthe amino acid residues having a smaller side chain volume areindependently selected from the group consisting of alanine (A), serine(S), threonine (T), valine (V).
 5. The antibody according to claim 3,wherein three amino acid residues are replaced with three amino residueshaving a smaller side chain volume, wherein the amino acid residueshaving a smaller side chain volume are independently selected from thegroup consisting of alanine (A), serine (S), threonine (T), valine (V).6. The antibody according to claim 2 wherein both CH3 domains arefurther altered by the introduction of a cysteine (C) as an amino acidin the same residue position of each CH3 domain.
 7. The antibodyaccording to claim 3 wherein both CH3 domains are further altered by theintroduction of a cysteine (C) as an amino acid in the same residueposition of each CH3 domain.
 8. The antibody according to claim 4wherein both CH3 domains are further altered by the introduction of acysteine (C) as an amino acid in the same residue position of each CH3domain.
 9. The antibody according to claim 1, wherein one of the CH3domains is replaced by a constant heavy chain domain CH1; and the otherCH3 domain is replaced by a constant light chain domain CL.
 10. A methodfor the preparation of an a bivalent, bispecific antibody according toclaim 1 comprising the steps of a) transforming a host cell with vectorscomprising nucleic acid molecules encoding the light chain and heavychain of an antibody specifically binding to a first antigen, andvectors comprising nucleic acid molecules encoding the light chain andheavy chain of an antibody specifically binding to a second antigen,wherein: within the light chain of the antibody which binds to saidsecond antigen, the variable light chain domain VL is replaced by thevariable heavy chain domain VH of said antibody and the constant lightchain domain CL is replaced by the constant heavy chain domain CH1 ofsaid antibody; and, within the heavy chain of the antibody which bindsto said second antigen, the variable heavy chain domain VH is replacedby the variable light chain domain VL of said antibody, and the constantheavy chain domain CH1 is replaced by the constant light chain domain CLof said antibody; b) culturing the host cell under conditions that allowsynthesis of said antibody molecule; and c) recovering said antibodymolecule from said culture.
 11. A host cell comprising: vectorscomprising nucleic acid molecules encoding the light chain and heavychain of an antibody specifically binding to a first antigen, andvectors comprising nucleic acid molecules encoding the light chain andheavy chain of an antibody specifically binding to a second antigen,wherein: within the light chain of the antibody which binds to saidsecond antigen, the variable light chain domain VL is replaced by thevariable heavy chain domain VH of said antibody and the constant lightchain domain CL is replaced by the constant heavy chain domain CH1 ofsaid antibody; and, within the heavy chain of the antibody which bindsto said second antigen, the variable heavy chain domain VH is replacedby the variable light chain domain VL of said antibody, and the constantheavy chain domain CH1 is replaced by the constant light chain domain CLof said antibody.
 12. A composition comprising a bivalent, bispecificantibody according to claim 1 and at least one pharmaceuticallyacceptable excipient.
 13. A composition comprising a bivalent,bispecific antibody according to claim 2 and at least onepharmaceutically acceptable excipient.
 14. A composition comprising abivalent, bispecific antibody according to claim 6 and at least onepharmaceutically acceptable excipient.
 15. A composition comprising abivalent, bispecific antibody according to claim 9 and at least onepharmaceutically acceptable excipient.